Nucleoids are prepared cell nuclei used to discern the structure and function of the nucleus. They have been used to characterize nuclear chromatin, nascent RNA, nuclear proteins, and nuclear structures, such as the nuclear matrix, lamina, and nuclear pore complex. Originally, the term "nucleoid" was used to describe the nuclear body within bacteria that contains the centrally located chromosomes. These bacterial nucleoids were isolated from spheroplasts using solutions containing 0.1 to 2.0 M salt and a chelating agent (1, 2). Subsequently, the term has come to include nucleoids from eukaryotes prepared similarly. Eukaryotic nucleoids have been isolated by gently lysing cells with 0.4 to 2.0 MNaCl, the chelating agent EDTA (133 mM), and the nonionic detergent Triton X-100 (0.67%, v/v). Cell lysis gently occurs over a 15-min period, and the nucleoids are enriched by sucrose gradient centrifugation (3). A "nucleoid cage" is formed by remnants of the cytoskeleton. The plasma membrane and cytoplasmic organelles are absent, but within the nucleoid cage is the nuclear cage. This inner nuclear cage consists of the nuclear envelope and the entire genome, although the histone proteins may be lost, depending on the salt concentrations used during lysis. Although intact, approximately 55% of the genome spills out of the nuclear material to form a shroud of intact chromatin loops that surrounds the nucleoid cage. The nuclear cage of the nucleoid contains residual nucleoli, nuclear particles, and fibrous aggregates that may be related to the chromosome scaffold described by Paulson and Laemmli (4).
When nucleoids are treated with low concentrations of ethidium bromide (5 |ig/mL), negative supercoils within the genomic DNA relax, and the DNA diffuses from the nucleoid to form a fluorescent halo around the nucleoid periphery. When the ethidium concentration is increased to 100 mg/mL, however, the halo of DNA is resorbed back into the nucleoid (5), because of the introduction of positive supercoils (6). If the DNA is nicked before adding excess ethidium bromide, it fails to retract back into the nucleoid, presumably because it is impossible to introduce positive supercoils. The observation that chromatin spools out and then back again as the DNA is positively supercoiled by ethidium bromide indicates that chromatin exists as closed looped domains. These early observations also suggested the presence of a nuclear matrix to which certain regions of the chromatin are anchored. Nucleoids were further used to demonstrate specific complexes within the nuclear matrix that are responsible for DNA replication and RNA transcription .
Experimental observations using nucleoids have received criticism in that artifactual associations between DNA or RNA with nuclear structures may arise due to the high salt conditions used in lysis. Jackson (7) provides arguments that such artifacts may be minimal: (1) the chromatin loops retain their size and the positions of genes on the loops remain constant; (2) preparation of nucleoids does not severely affect the apparent specificity of nascent RNA association with the nuclear matrix (8); (3) DNA, RNA, or ribonucleoprotein particles added before or after nucleoid purification in the presence of 2 M NaCl fail to associate with the nuclear cage (8); (4) no gross redistribution of DNA and RNA occurs during nucleoid preparation. Regardless of criticisms, nucleoids provided initial sources for studying nuclear matrices that many now accept as the structural support for nascent DNA replication, RNA transcription, pre-mRNA splicing, and RNA transport (9) (see Nuclear Matrix).
