Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 72
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In one type of large-fragment electrophoresis, called pulsedfield gel electrophoresis (PFGE), the apparatus is similar to that for conventional electrophoresis, but theorientation of the electric field is changed periodically; the improved separation of large DNA moleculesapparently results from the additional time it takes for large molecules to reorient themselves when the orientationof the electric field is changed. Figure 6.6 illustrates the separation of the 16 chromosomes from yeast by means ofPFGE. The chromosomes range in size from approximately 200 kb to 2.2 Mb. Electrophoretic separation yields avisual demonstration that each of the chromosomes contains a single DNA molecule that runs continuouslythroughout its length. Using the methods discussed in Section 5.7, we can determine the identity of eachchromosomal band in the gel by hybridization of a Southern blot with probes for genes known to map to aparticular chromosome.The Nucleosome Is the Basic Structural Unit of ChromatinThe DNA of all eukaryotic chromosomes is associated with numerous protein molecules in a stable, orderedaggregate called chromatin.
Some of the proteins present in chromatin determine chromosome structure and thechanges in structure that occur during the division cycle of the cell. Other chromatin proteins appear to playimportant roles in regulating chromosome functions.Page 229Figure 6.6Separation of the 16 chromosomes of yeast by pulsed-field gel electrophoresis,in which there is regular change in the orientationof the electric field. Bands for each of the chromosomes are clearly visible.They range in size from 200 kb to 2.2 Mb.[Courtesy of BioRad Laboratories, Hercules, California.]Nucleosome Core ParticlesThe simplest form of chromatin is present in nondividing eukaryotic cells, when chromosomes are not sufficientlycondensed to be visible by light microscopy.
Chromatin isolated from such cells is a complex aggregate of DNAand proteins. The major class of proteins comprises the histone proteins. Histones are largely responsible for thestructure of chromatin. Five major types—H1, H2A, H2B, H3, and H4—are present in the chromatin of nearly alleukaryotes in amounts about equal in mass to that of the DNA. Histones are small proteins (100–200 amino acids)that differ from most other proteins in that from 20 to 30 percent of the amino acids are lysine and arginine, both ofwhich have a positive charge.
(Only a few percent of the amino acids of a typical protein are lysine and arginine.)The positive charges enable histone molecules to bind to DNA, primarily by electrostatic attraction to thenegatively charged phosphate groups in the sugar-phosphate back-bone of DNA. Placing chromatin in a solutionwith a high salt concentration (for example, 2 molar NaCl) to eliminate the electrostatic attraction causes thehistones to dissociate from the DNA. Histones also bind tightly to each other; both DNA-histone and histonehistone binding are important for chromatin structure.The histone molecules from different organisms are remarkably similar, with the exception of H1. In fact, theamino acid sequences of H3 molecules from widely different species are almost identical.
For example, thesequences of H3 of cow chromatin and pea chromatin differ by only 4 of 135 amino acids. The H4 proteins of allorganisms also are quite similar; cow and pea H4 differ by only 2 of 102 amino acids. There are few other proteinswhose amino acid sequences vary so little from one species to the next. When the variation between organisms isvery small, we say that the sequence is highly conserved.
The extraordinary conservation in histone compositionthrough hundreds of millionsPage 230of years of evolutionary divergence is consistent with the important role of these proteins in the structuralorganization of eukaryotic chromosomes.In the electron microscope, chromatin resembles a regularly beaded thread (Figure 6.7). The bead-like units inchromatin are called nucleosomes. The organization of the nucleosomes in chromatin is illustrated in Figure 6.8A.Each unit has a definite composition, consisting of two molecules each of H2A, H2B, H3, and H4, a segment ofDNA containing about 200 nucleotide pairs, and one molecule of histone H1.
The complex of two subunits each ofH2A, H2B, H3, and H4, as well as part of the DNA, forms each ''bead," and the remaining DNA bridges betweenthe beads. Histone H1 also appears to play a role in bridging between the beads, but it is not shown in Figure 6.8A.Brief treatment of chromatin with certain DNases (for example, micrococcal nuclease from the bacteriumStaphylococcus aureus) yields a collection of small particles of quite uniform size consisting only of histones andDNA (Figure 6.9). The DNA fragments in these particles are of lengths equal to about 200 nucleotide pairs orsmall multiples of that unit size (the precise size varies with species and tissue). These particles result fromcleavage of the linker DNA segments between the beads (Figure 6.8B).
More extensive treatment with DNaseresults in loss of the H1 histone and digestion of all the DNA except that protected by the histones in the bead. Theresulting structure, called a core particle, consists of an octamer of pairs of H2A, H2B, H3, and H4, around whichthe remaining DNA, approximately 145 base pairs, is wound in about one and three-fourths turns (Figure 6.8B).Each nucleosome is composed of a core particle, additional DNA called linker DNA that links adjacent coreparticles (the linker DNA is removed by extensive nuclease digestion), and one molecule of H1; the H1 binds tothe histone octamer and to the linker DNA, causing the linkers extending from both sides of the core particle tocross and draw nearer to the octamer, though some of the linker DNA does not come into contact with anyhistones. The size of the linker ranges from 20 to 100 nucleotide pairs for different species and even in differentcell types in the same organism (200 - 145 = 55 nucleotide pairs is usually considered an average size).
Little isknown about the structure of the linker DNA or about whether it has a special genetic function, and the cause of thevariation in its length is also unknown.The Arrangement of Chromatin Fibers in a ChromosomeThe DNA molecule of a chromosome is folded and folded again in such a way that it is convenient to think ofchromosomes as having several hierarchical levels of organization, each responsible for a particular degree ofshortening of the enormously long strand (Figure 6.10). Assembly of DNA and histones can be considered the firstlevel—namely, a sevenfold reduction in length of the DNA and the formation of a beaded flexible fiber 110 Å (11nm) wide (Figure 6.10B), roughly five times the width of free DNA (Figure 6.10A).
The structure of chromatinvaries with the concentration of salts, and the 110 Å fiber is present only when the salt concentration is quite low.If the salt concentration is increased slightly, then the fiber becomes shortened somewhat by forming a zigzagarrangement of closely spaced beads between which the linking DNA is no longer visible in electron micrographs.IfFigure 6.7Dark-field electron micrograph of chromatin showing the beaded structure atlow salt concentration. The beads have diameters of about 100 Å.[Courtesy of Ada Olins.]Page 231Figure 6.8(A) Organization of nucleosomes. The DNA molecule is wound one and three-fourths turns around a histone octamercalled the core particle.
If Hl were present, it would bind to the octamer surface and to the linkers, causing the linkers tocross. (B) Effect of treatment with micrococcal nuclease. Brief treatment cleaves the DNA between the nucleosomes andresults in core particles associated with histone Hl and approximately 200 base pairs of DNA. More extensivetreatment results in loss of Hl and digestion of all but 145 base pairs of DNA in intimate contact with each core particle.Figure 6.9Electron micrograph of nucleosomemonomers.[Courtesy of Ada Olins.]Page 232figure 6.10Various stages in the condensation of DNA (A) and chromatin (B through E) in forming a metaphase chromosome (F).The dimensions indicate known sizes of intermediates, but the detailed structures are hypothetical.Page 233the salt concentration is further increased to that present in living cells, then a second level of compaction occurs:the organization of the 110 Å nucleosome fiber into a shorter, thicker fiber with an average diameter ranging from300 to 350 Å, called the 30 nm fiber (Figure 6.10C).
In forming this structure, the 110 Å fiber apparently coils in asomewhat irregular left-handed superhelix or solenoidal supercoil with six nucleosomes per turn (Figure 6.11). It isbelieved that most intracellular chromatin has the solenoidal supercoiled configuration.The final level of organization is that in which the 30 nm fiber condenses into a chromatid of the compactmetaphase chromosome (Figure 6.10D through F). Little is known about this process other than that it seems toproceed in stages.
In electron micrographs of isolated metaphase chromosomes from which histones have beenremoved, the partly unfolded DNA has the form of an enormous number of loops that seem to extend from acentral core, or scaffold, composed of nonhistone chromosomal proteins (Figure 6.12). Electron microscopicstudies of chromosome condensation in mitosis and meiosis suggest that the scaffold extends along the chromatidand that the 30 nm fiber becomes arranged into a helix of loops radiating from the scaffold.