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Lecture 11, part 1 of 2
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October 10, 2017



F. The nucleus is organized for carrying out transcription and replication.
1. Decondensed chromosomes in the interphase nucleus occupy discrete territories.

2. A disk-shaped nucleus accommodates nuclear/cytoplasmic transport.
G.The large scale structure of chromosomes
1.Evidence that the eukaryotic chromosome is a single linear DNA molecule.
a. Replication in the presence of bromodeoxyuridine
b. Pulsed-field electrophoresis of yeast chromosomal DNA
c. Complete nucleotide sequence of the Yeast genome
2. Higher-level coiling
a. SEM Studies of Human Chromosomes
H. Summary

Last time, we looked at all the organizational features necessary to handle gene expression in chromatin domains. Today, we need to look at the higher-level structural features required for transcription, replication and chromosome segregation.

F. The nucleus is organized for carrying out transcription and replication.

1. Decondensed chromosomes in the interphase nucleus occupy discrete territories.

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.

Andreas Bolzer, Gregor Kreth, Irina Solovei, Daniela Koehler, Kaan Saracoglu, Christine Fauth, Stefan Müller, Roland Eils, Christoph Cremer, Michael R. Speicher, Thomas Cremer - Bolzer et al., (2005) Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei and Prometaphase Rosettes. PLoS Biol 3(5): e157 DOI: 10.1371/journal.pbio.0030157, part of Figure 1.

Top: FISH (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.

2. A disk-shaped nucleus accommodates nuclear/cytoplasmic transport.

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.

G. The large scale structure of chromosomes

1. The eukaryotic chromosome is a single linear DNA molecule.

We have been discussing domains of chromatin as functional units in gene expression, some up to 150kb in length. But we haven't yet addressed the question, "does a chromosome have many molecules of DNA, or only one?" There are three lines of evidence which demonstrate that eukaryotic chromosomes are single linear DNA molecules.
This experiment 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

Alternative site: NCBI

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