Birefringence and DNA Condensation of Liquid Crystalline Chromosomes

Abstract

DNA can self-assemble in vitro into several liquid crystalline phases at high concentrations. The largest known genomes are encoded by the cholesteric liquid crystalline chromosomes (LCCs) of the dinoflagellates, a diverse group of protists related to the malarial parasites. Very little is known about how the liquid crystalline packaging strategy is employed to organize these genomes, the largest among living eukaryotes-up to 80 times the size of the human genome. Comparative measurements using a semiautomatic polarizing microscope demonstrated that there is a large variation in the birefringence, an optical property of anisotropic materials, of the chromosomes from different dinoflagellate species, despite their apparently similar ultrastructural patterns of bands and arches. There is a large variation in the chromosomal arrangements in the nuclei and individual karyotypes. Our data suggest that both macroscopic and ultrastructural arrangements affect the apparent birefringence of the liquid crystalline chromosomes. Positive correlations are demonstrated for the first time between the level of absolute retardance and both the DNA content and the observed helical pitch measured from transmission electron microscopy (TEM) photomicrographs. Experiments that induced disassembly of the chromosomes revealed multiple orders of organization in the dinoflagellate chromosomes. With the low protein-to-DNA ratio, we propose that a highly regulated use of entropy-driven force must be involved in the assembly of these LCCs. Knowledge of the mechanism of packaging and arranging these largest known DNAs into different shapes and different formats in the nuclei would be of great value in the use of DNA as nanostructural material.DNA molecules are the indispensable genetic material of every organism. Apart from having the encoding power of a 4-base double-stranded polymer, DNA also has an enormous capacity to be condensed. It is probably this ability that allowed the evolution of increasing genome sizes in the eukaryotes. Histone-mediated nucleosome-based chromatin is the prevailing method for DNA packaging, but it is not the only way that DNA is condensed in the eukaryotes. Highly compact liquid crystalline DNA has been reported in several animal sperm nuclei (11, 28) and was also found in the nucleosomeless liquid crystalline chromosomes (LCCs) of dinoflagellates. Neither animal sperm nuclei nor dinoflagellate nuclei employ histones. In fact, the histone core octamer may hinder the attainment of ultrahigh levels of condensation due to its restrictive volume. In the sperm model, protamine is a sperm-specific DNA-binding protein (ϳ7 kDa) that is an order of magnitude smaller than the histone core octamer, and it adopts a structural role in the organization of the male gametic genome (1, 3). It is this very reduction in the protein-to-DNA ratio that may well enable the high DNA compaction into a liquid crystalline state. The dinoflagellate chromosomes are known to have a proteinto-DNA ratio even smaller than those of the prokaryo...

The analysis of microscopy imaging on liquid crystalline components of the cell nucleus

“…After Fourier analysis, the data obtained by Metripol microscopy includes phase shift information for the extinction angle and the amplitude of retardation with the application of continuously rotating polarizer [36]. If all intranuclear chromosomes have similar local chromosomal DNA concentration, then all chromosomes may have similar birefringence properties [11]. However, although all intranuclear chromosomes exhibit equal birefringence magnitudes, some regions exhibit much lower birefringent intensities.…”

“…The overlapping effects from different directions of chromatin orientation may also contribute to such anisotropic distributions. As the light passes through the liquid crystalline nuclei, the change in the angle of vibration direction of the polarized light will generate difference for highly compacted chromatins (Figure 1(c)) [11].…”

“…The radially distributed intranuclear chromosomes are accompanied by certain regions that exhibit much lower magnitudes of birefringence ( Figure 1(b)). The colors within the orientation angle micrograph indicate the chromosomal alignment from purple (horizontal, 0˚) to red (horizontal, 180˚), and the orientation is related to the angle between the long axis of the chromosome and the direction of the chromatin filament fiber alignment (Figure 1(c)) [11].…”

“…High-resolution bioimaging of the highly condensed intranuclear organization is a challenge with current microscopy techniques because conventional light microscopy has a resolution limit of approximately half the wavelength of light, which is approximately one-fourth the diameter of a typical chromosome, and TEM micrographs of ultrathin sections of chromosomes lose information on the nuclear spatial architecture [10]. Therefore, quantitative determination of the local chromosomal DNA concentration is not easy because it is difficult to obtain the precise volume fraction of chromosome-occupied based on neither optical microscopy nor electronic microscopy [7,8,11]. Consequently, relatively little is known about the biophysical biological aspects of intranuclear genome condensation in macromolecular crowded environment [5].…”

Dinoflagellate tandem array gene transcripts are highly conserved and not polycistronic

“…Dinoflagellates also display several unusual cytological and biochemical features. For example, their chromosomes remain permanently condensed throughout the cell cycle (5) and electron microscopy of dinoflagellate nuclei shows a whorled structure termed a cholesteric liquid crystal (6). It seems likely that this unusual nuclear organization may impose restrictions on DNA replication and transcription, yet details of these processes are still unknown.…”