|last page||PLNT3140 Introductory Cytogenetics
Lecture 11, part 1 of 2
F. The nucleus is organized for carrying out transcription and replication.
1. Decondensed chromosomes in the interphase nucleus occupy discrete territories.G.The large scale structure of chromosomes
2. A disk-shaped nucleus accommodates nuclear/cytoplasmic transport.
1.Evidence that the eukaryotic chromosome is a single linear DNA molecule.H. Summary
a. Replication in the presence of bromodeoxyuridine2. Higher-level coiling
b. Pulsed-field electrophoresis of yeast chromosomal DNA
c. Complete nucleotide sequence of the Yeast genome
a. SEM Studies of Human Chromosomes
|Chromosome painting demonstrates that
chromosomes occupy discrete volumes within the interphase
nucleus. There are referred to as "chromosome territories".
Note that the image at right is based on a 2-dimensional slice through a 3-dimensional nucleus. Not all copies of all chromosomes can be seen in any given plane.
(Fluorescence in situ hybridization) labeling of all
24 different human chromosomes (1 - 22, X, and Y) in
a fibroblast nucleus, each with a different
combination of in total seven fluorochromes. Shown is
a mid-plane of a deconvoluted image stack which was
recorded by wide-field microscopy. Bottom: False color
representation of all chromosome territories visible
in this mid-section after computer classification.
"Painting" of chromosomes with flourescently-tagged transcription factors allows real-time visualization of the location of specific chromosomal sites.
Marshall WF, Straight A, Marko JF, Swedlow J, Dernburg A, Belmont A, Murray AW, Agard DA, and Sedat JW. 1997. Interphase chromosomes undergo constrained diffusional motion in living cells. Current Biology 7,930-939.
Researchers in Dr. John Sedat's lab at UCSF wanted to determine whether specific loci for a given homologous pair of chromosomes were physically associated during interphase. This required an imaginative approach. Yeast was transformed with the E. coli lac operator sequence, which is specifically bound by the lac repressor protein. Next, the same yeast strain was transformed with a chimeric gene in which the Green Fluroescent Protein (GFP) was added to the amino terminus of the E. coli lac repressor gene. The chimeric gene was inserted at random into a different chromosomal site. Since the chimeric lac repressor/GFP protein will only bind to the lac repressor sequence, a diploid yeast nucleus should only have two binding sites, one for each homologous locus.
Collings DA et al. (2000) Plant nuclei can contain extensive grooves and invaginations. Plant Cell 12: 2425-2439.
|A - Serial sections through onion
epidermal nuclei, in which DNA has been labeled using DAPI
dye. DNA is visualized by UV fluorescence.
B. - Light microscopic images of sections shown in A.
C - 3D reconstruction of surface based on DAPI fluorescence. Nc = nucleoli; G = groove; arrow shows an invagination.
from Figure 1.
Copyright © 2000 by the American Society of Plant Biologists
Recalling that the
nucleus is thought to be a specialized structure formed from the
endoplasmic reticulum, what is the importance of these findings?
That is, how might a lens shape with a convoluted surface
function more efficiently than a rigid spherical shape?
A sphere is the
geometrical shape that minimizes surface area per unit volume.
Both the lens shape and the channels, invaginations and groves
would contribute to increasing the surface area. Since the
nuclear envelope controls traffic of macromolecules into and out
of the cell, that traffic can be carried more efficiently with a
large surface area. In particular, grooves and invaginations
extending deep into the nucleus ensure that no part of the
nucleus is far from the nuclear membrane. This will minimize the
time required for a transcript to exit the nucleus, or for a
protein to enter, and find its way to a chromosome.
demonstrates that each chromatid, and hence the
chromosome, is a single DNA molecule. If this were not
so, then we would simply see an even distribution of dye
in both chromatids, and a gradual dilution of BUdr
(dark) in subsequent cell generations. Instead,
we see a discrete partitioning of the BUdr into
one or the other of the sister chromatids, which is
consistent with the idea that each chromatid contains one
old strand and one newly-replicated strand.
In some chromosomes, dye has been partitioned completely to one chromatid or the other (circled). In other chromosomes, sister-chromatid exchange is evidenced by a checkered-pattern, in which dye abruptly shifts from one chromatid to the other (arrow)
image from Kimball's Biology Pages
(Saccharomyces cerevisiae ) cells were embedded
in agarose, and the agarose plugs treated with enzymes
to degrade the cell walls. Agarose plugs are then
loaded into the wells of a pulsed-field gel.
All 15 yeast chromosomes can be resolved on this gel.
Image displayed from http://bio3400.nicerweb.com/Locked/media/ch19/pulsed-field-gel-electrophoresis.html
ii) Linear molecules correspond in length to estimated lengths of chromosomes.
Yeast was the first eukaryotic genome to be completely sequenced. The first complete yeast chromosomal sequence was from chromosome III.
Database entry: 315339bp
182 ORFs > 100bp (mostly unknown genes)
The complete yeast genome can be searched and browsed at the Saccharomyces Genome Database (SGD) at Stanford: http://www.yeastgenome.org/
Alternative site: NCBI
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