Index
It is the region that contains DNA in prokaryotes.
Microorganisms have provided robust systems for understanding the basic principles of chromosome organization, dynamics, and function, beginning with the replicon hypothesis.
Over the past decade, increasingly sophisticated image analysis from physical perspectives has been added to the more traditional tools of biochemistry, genetics, and molecular biology.
A significant challenge for current research is integrating these considerations to provide a more in-depth and unified view.
The nucleoid
The nucleoid is the space within a prokaryotic cell, called a genophore, where genetic information is located. Prokaryotes are divided into bacteria and archaea, single-celled organisms that do not contain membrane-bound organelles.
The nucleoid, then, does not have a membrane around it either. It adheres to the cell membrane and is in immediate contact with the cytoplasm.
The nucleoid is also not uniform in shape and does not have a specific size. However, we can still distinguish it from the rest of the cell and identify it under a light microscope.
The nucleoid is mainly composed of multiple copies packed into a continuous strand of DNA, with the addition of some RNA and proteins. DNA in prokaryotes is double-stranded and generally takes a circular shape.
Note that DNA can sometimes be found in other regions outside of the nucleoid. To put things in perspective, we can look at the eukaryotic counterpart of the nucleoid.
Like plants and animals, Eukaryotes have a nucleus that houses their genetic material, with a surrounding double membrane, or what we call the nuclear envelope.
This membrane separates the contents of the nucleus from the cytoplasm. As in prokaryotes, eukaryotic DNA is also double-stranded.
In bacteria
A significant advance in recent years has been the appreciation that the circular DNA of the bacterial chromosome, called “nucleoid,” is a discrete and well-defined physical object.
Fresh fluorescence images of living cells and fixed cell chromosome capture analysis reveal discrete shapes with defined longitudinal substructure.
These findings confirm and extend previous image analysis findings and put models in which the nucleoid comprises a randomly oriented fiber, either linear or topologically branched, that fills the available cell space to rest.
Nucleosides are prepared from isolated plastids by solubilizing the envelope and thylakoid membranes. Various nonionic detergents have been used, as well as mixed density gradient media.
However, the most significant improvement was using the so-called “TAN” tampon invented by the Kuroiwa group. “TAN” was the name of a researcher who first used it in his laboratory.
The TAN buffer consists of 0.5 M sucrose, 1.2 mM spermidine, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride, and 20 mM tris aminomethane hydrochloride (Tris HCL) (pH 7.5). Sucrose and spermidine are highly effective in stabilizing the compact structure of nucleoids.
Low ionic strength and chelation of divalent cations with ethylenediaminetetraacetic acid are essential to maintain nucleoids in a highly condensed state.
The addition of sodium chloride (NaCl) at a concentration above 0.2 M or several millimolar magnesium chlorides (MgCl2) releases major DNA-binding proteins from nucleosides.
Spermidine is a member of natural polyamines, which are present in chloroplasts and bacterial or eukaryotic cells and are essential for maintaining proper gene expression activity.
However, spermidine should not be added to the buffer during the isolation of plastids by the Percoll density gradient, as it binds to the negatively charged surface of Percoll.
A simple and convenient method for preparing nucleoids from plastids is to:
Solubilization of plastids with Nonidet P-40, low-speed centrifugation, and filtration through a membrane filter to remove bulk contaminants (starch, cell nucleus fragments, cuticles, and other insoluble materials), and nucleoid sedimentation by high-speed centrifugation.
The use of density gradient centrifugation through metrizamide or sucrose effectively purifies nucleoids to some extent. Still, the density gradient’s effectiveness is limited because nucleoids are highly heterogeneous concerning buoyancy density.
Unfortunately, even the purest of currently available plastid preparations contain several hydrophobic proteins that are not believed to be actual constituents of nucleosides.
Although isolated nucleoid preparations appear very pure upon inspection under a fluorescence microscope after staining with fluorochrome DAPI (4 ‘, 6-diamidino-2-phenylindole), several hydrophobic proteins originating from various cell membranes adhere to the preparation. Nucleoid.
As demonstrated by the microsequencing of representative protein bands of the nucleoids.
This may be why a comprehensive proteomic analysis of plastid nucleosides has not been performed. Currently, the critical components are identified one by one by molecular biological and biochemical means.
“reconstitution” experiments identify various DNA-binding proteins. A protein overexpressed in Escherichia coli cells and highly purified from them is mixed with the purified DNA to see the formation of particulate structures that resemble cells. Nucleosides under microscopy.
Another method is to use DAPI as a fluorescent probe to monitor DNA condensation. This method is based on changes in fluorescence quenching caused by condensation of DNA and fluorochrome.
The effect of the addition of nucleoid proteins, which are overexpressed in E. coli cells, on the transcriptional activity of nucleoids is a good criterion for identifying functionally important components of nucleoids, an approach that complements biochemical analysis.
The protein composition of nucleoids isolated from several plants and organs has been reported.
Changes in protein composition or transcription activity during the development of chloroplasts, chromoplasts, proplastides, and amyloplasts are described.
The nucleosides of etioplasts and chloroplasts in cucumbers and peas have been compared.
The protein composition is primarily identical in cucumber cotyledon etioplasts and chloroplasts, while the pea leaf etioplast and chloroplast nucleoplasm contained qualitatively and quantitatively different proteins.
The etioplasts of cucumber cotyledons are large and contain highly developed prolamellar bodies, ready to become chloroplasts shortly after illumination (about 3 to 6 h).
While the etioplasts of pea leaves are small and begin to develop for a long time (more than 24 h) after the onset of illumination.
This suggests that the composition of the nucleoids is determined not only by the state of greening but by the state of global development of the plastid.
Nuclear structure and chromosome replication
The central feature of bacterial nucleic acid metabolism is the nucleoid. This cytoplasmic region is so densely packed with nuclear material that it excludes ribosomes and can be easily seen on electron micrographs.
The nucleoid generally occupies about 10% of the cell volume. The nucleoid is not enclosed by a nuclear membrane found in eukaryotes, which has significant consequences for the organism.
The nucleoid comprises approximately 60% DNA, 30% RNA, and 10% proteins. The chromosome, made up of DNA, is the predominant molecule in the nucleoid.
Typically, there is a chromosome approximately 1 mm in length, with a typical prokaryotic genome size of 1.9 to 6.3 x 106 base pairs.
This DNA is a double-stranded, circular, covalently closed (usually) supercoiled molecule.
Several species of prokaryotic microorganisms have linear chromosomes, and some species have two or three chromosomes.
Considering the large size of the chromosome, it is not surprising that it must be compacted to fit in the bacterial cell. Since there is no nuclear membrane, RNA and protein keep the nuclear structure compact.
The chromosome exists as 50–100 separately coiled domains that can be individually relaxed for gene replication, repair, and expression.
Supercoiling is maintained by topoisomerase enzymes, such as DNA gyrase (which supports negative supercoiling and relaxes).
Other proteins essential for maintaining nucleoid structure are the non-specific HU DNA-binding proteins and histone-like nucleolus structuring (H-NS), and the IHF (host integrating factor) protein which has sequence binding sites of Specific DNA.
Chromosome replication occurs by the same universal principles guiding images in all biological systems.
DNA synthesis is semi-conservative and always occurs in the five ′ to 3 ′ directions.
DNA synthesis is semi-continuous, and DNA polymerase requires a template and a primer.
There are three DNA polymerase enzymes in most bacteria, with the holoenzyme DNA polymerase III complex being responsible for chromosome replication.
DNA synthesis always begins at the same site called the origin of replication and is bidirectional from that origin.
In addition to the polymerase complex, there are a variety of other enzymes that contribute to initiation and synthesis. Some of these enzymes can be targets for antibiotics.
The nucleoid is self-adhesive.
Several lines of evidence reveal a solid tendency to the general coalescence of chromosomal material, that is, that the nucleoid is “self-adherent.”
The images reveal that virtually all chromosomal DNA is part of the nucleoid shape.
Furthermore, during the replication/segregation process, it can sometimes be observed that elongation involves lobes of the newly replicated material protruding, implying an intrinsic dynamic tendency for coalescence in elongated shapes.
Finally, self-adherence is implicit in the finding that individual loci and pairs of loci tend to have pretty fixed positions relative to each other in the resting nucleosides (G1).
Radial, but not longitudinal, confinement
Non-septate cells exhibit discrete nucleoside chains in the absence of intercellular boundaries; furthermore, the G1 nucleoid does not always extend to the end of the cell.
Therefore, the nucleoid is a discrete object in the absence of “longitudinal confinement.”
In contrast, the nucleoid touches the inner periphery of the cell in the radial dimension.
Since the shape tends to be helically curved, this contact is not uniform but reflects the winding path.
One implication of this configuration is that the nucleoid tends to define a complementary helical space around the cell’s periphery.
Regardless of the molecular linkages between the nucleoid and the inner cell membrane, it also appears that the nucleoid tends to “push” the periphery of the cell out, that is, that the shape is “radially confined.”
The confinement of the nucleoid in the radial dimension figures prominently in various aspects of the organization, arrangement, and function of chromosomes.
At “G1,” a curved ellipsoidal shape with underlying longitudinal duality
The nucleoid before replication (“G1”), as defined in E.coli and C. crescentus, tends to be thinner at the ends than in the middle, that is, be ellipsoidal and deform into a helix with a smooth curve.
The curvature does not necessarily have a specific ability, which implies that the essential characteristic is the curvature itself. Furthermore, nucleoid DNA is denser centrally than radially.
Behind this shape, DNA tends to organize itself into a pair of parallel beams that extend longitudinally along the length of the nucleoid and rotate smoothly relative to each other to give the nucleoid shape a helical, smoothly curved shape.
In C. crescentus, the two packages correspond to the left and right replicators.
In some circumstances, including late stages of the cell cycle, the nucleoid appears as an open ring and well-separated bundle pairs.
Longitudinal confinement could potentially influence form at these stages.
Dual Radial Arrays of Plectonemic Loops
Chromosome capture analysis in C. crescentus suggests that, in this organism, the longitudinal duality probably reflects two similar objects arranged in a ‘bottle brush’ shape, each comprising a radial series of plectonemic loops.
Each loop would be ~ 15kb long with a ~ 100kb super arrangement. A similar underlying organization likely explains the duality of E.coli.
If so, the two characteristics identified could reconcile previous observations in E.coli that differently defined a topologically supercoiled domain by ~ 50–100kb versus environments of ~ 10–15kb).
Domain differentiation
For most Enterobacteriaceae and Vibrionaceae, the terminal region is highly structured through a dedicated protein, MatP, a plectonema-binding protein with a preferred binding sequence whose domain location is specifically nucleated.
The origin is also embedded in a structured region, as shown in B.subtils and E.coli. In B. subtle, this structure is created by condensin and nucleated by ParB. The origin and termination domains also occur in P.aeruginosa.
For E.coli, domain differentiation of interstitial regions has also been suggested.
The experimental definitions of these regions can reflect both the segregation process and the physical transitions of different macrosegments of the genome.
Dynamic
The time-lapse images have defined local dynamic movements and revealed global emotional behaviors.
Movement of loci on short time scales
The local movement of a fluorescently labeled chromosomal locus is defined by plotting its mean square displacement (DCM) versus elapsed observation time.
In a log-log plot, simple diffusive motion provides a linear relationship with a slope of 1.
For the chromosomal loci in bacteria, the relationship is linear but with a slope of less than 1, implying a “sub-diffusive” movement.
This behavior is due to the viscoelastic nature of the crowded nucleoid: as a locus moves, it pushes against the constraints of the environment, which go back, thus preventing movement.
The alternative explanations, that is, the adherence of the place to the characteristics found or impediments of the fixed obstacles, are not compatible with the observed patterns.
An important subcomponent of the apparent diffusion coefficient is non-thermal, with ATP-dependent processes promoting agitation.
So far, no single dominant motor protein has been identified, with DNA gyrase, cell wall biosynthesis, and the MreB cytoskeleton excluded as significant players and RNA polymerase having only a minor role.
A monitored locus may occasionally exhibit rapid movements exhibiting “near ballistic” dynamics, involving active translocation machinery or stress-relaxation effects.
Such movements occur for loci throughout the genome but differently with different genomic coordinates.
These movements do not strictly correlate with DNA replication; they tend to occur along the length of the nucleoid and sometimes accompany chromosome segregation transitions.
Two types of global dynamics at the nucleotide level
Whole nucleoid images in E. coli reveal two dynamic behaviors that involve the entire nucleoid.
These two behaviors come into play on different time scales. In no case is the underlying mechanism known.
In both cases, the elimination of tethers between segments (mediated by proteins and topological, lengthwise, and between sisters) is proposed to perform critical functions, making the nucleoid more “fluid.”
Fluency would facilitate the local movements required for various chromosomal processes, including, for example, the displacement of transcribed regions to move to the nuclear periphery for translation, as well as the dynamics of replication, sister segregation, and organization.
Longitudinal density waves
The total nucleoside density fluctuates along the length of the nucleoid with a periodicity of one to two minutes, probably throughout the cell cycle, with a net displacement of approximately 5% of the nucleoside material every 5 s.
These waves are proposed to promote the internal mobility of the nucleoid by promoting the loss of ties or entanglements between segments that would otherwise create a gel.
Such a role would be analogous to that suspected for the back and forth movements of meiotic prophase chromosomes in correlation with the removal of unwanted tangles created during chromosome pairing.
Cyclic nucleoid extension and shortening
The length of the cell increases monotonically during growth. In contrast, the size of the nucleosides varies discontinuously in a cyclic pattern.
In each cycle, five minutes of nucleoid shortening is followed by 20 minutes of lengthening.
The elongation rate increases from ~ 10min to a maximum and then decreases, finally becoming negative as shortening occurs, followed by the next phase of elongation.
These kinetics are surprisingly consonant with the accumulation, release, and dissipation of viscoelastic mechanical stress, which implies the existence of automated stress cycles.
So far, these cycles are documented for a period that includes but extends well beyond the period of DNA replication, with indications that it also occurs in G1.
Such cycles could comprise a primordial cell cycle that governs the program of chromosomal events and its link to the process of cell division.
These global cycles are also implicated explicitly in sister segregation.
It is proposed that random tethers between segments prevent the formation of a regular nucleolus organization (e.g., radial loop arrays), which tends to make the nucleoid larger and less elongated (i.e., broader and shorter ) in a high energy state (mechanical stress).
Eventually, the stress level will become high enough to cause the release of the restraints, thus allowing stress relief from lengthening.
This effect would be concomitantly blocked by the organization’s development (radial loop).
At first glance, it may seem counterintuitive that the expansion provided by nucleotide dynamics could be biologically valuable, as it is commonly accepted that chromosome compaction is a critical element of chromosome segregation and organization.
However, several scenarios can be imagined in which transient decay could help.
For example, one must remember that global condensation will not separate sisters and that, specifically, only condensation of length can drive segregation.
A general proposition is that such sister-specific condensation (generated, for example, by supercoiling) will be affected or at least slowed down by molecular cross-links.
Therefore, a local nucleoid expansion could decrease the number of cross-links, subsequently allowing a more efficient longitudinal condensation.
More generally, there is general agreement that the underlying organization of DNA within the nucleoid is relatively regular.
Molecular events, particularly transcription, will disrupt that organization, and those disruptions can be restricted and permanent by molecular cross-links.
Transient expansion waves could again suppress these crosslinks, allowing a reorganization of the nucleoid in its “ideal” state.
Nucleoid function
The nucleoid is essential to control cell activity and reproduction. It is where DNA transcription and replication take place.
Within it, we can expect to find enzymes that serve as biological catalysts and help with replication, as well as other proteins that have different functional and structural roles, such as assisting DNA formation, facilitating cell growth, and regulating the genetic material of the cell.
Conclusion
The dynamics of prokaryotic and eukaryotic chromosomes have always been predicted to be fundamentally similar.
Recent observations support this proposition and clarify the nature of the relationships.
Chromosomes tend to be discrete and coherent units in bacteria, as is well known for eukaryotes.
Both cell types are similar in the elastic environment that governs short-term locus mobility and its dependence on ATP (enzyme).
So is the observation that individual loci undergo ballistic movements. Chromosomes have a fundamental underlying organization that involves a radial series of loops with higher-order organization tracks.
Furthermore, in both cases, the central “backbone” defined by the loop bases and associated proteins includes condensin, AT-rich sequences, and architectural proteins that bind to these sequences.
The back and forth oscillations of the longitudinal density waves observed in E. coli have direct parallels in mammalian G1 nuclei and meiotic prophase.
The global cycles of contraction/extension observed in E. coli appear to be very parallel to the global processes of chromosome expansion/contraction observed in the meiotic and mitotic programs of the eukaryotic programs, which are also related to the individualization of the chromatid period. Sisters.
