Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 73
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Details are not knownabout the additional folding that is required of the fiber in each loop to produce the fully condensed metaphasechromosome.The genetic significance of the compaction of DNA and protein into chromatin and ultimately into thechromosome is that it greatly facilitates the movement of the genetic material during nuclear division. Relative to afully extended DNA molecule, the length of a metaphase chromosome is reduced by a factor of approximately 104as a result of chromosome condensation.
Without chromosome condensation, the chromosomes would become soentangledFigure 6.11(A) Electron micrograph of the 30 nm component of mouse metaphasechromosomes. (B) A proposed solenoidal model of chromatin. The DNA(blue-gray) is wound around each nucleosome. It is unlikely that thereal structure is so regular.[Electron micrograph courtesy of Barbara Hamkalo; drawing after J.
T. Finchand A. Klug. 1976. Proc. Nat. Acad. Sci.USA, 73: 1900.]Figure 6.12Electron micrograph of a partially disrupted anaphase chromosome ofthe milkweed bug Oncopeltus fasciatus, showing multiple loops of 30-nmchromatin at the periphery.[From V. Foe, H. Forrest, L. Wilkinson, and C. Laird. 1982. InsectUltrastructure, 1:222.]Page 234that there would be many more abnormalities in the distribution of genetic material into daughter cells.6.5—Polytene ChromosomesA typical eukaryotic chromosome contains only a single DNA molecule.
However, in the nuclei of cells of thesalivary glands and certain other tissues of the larvae of Drosophila and other two-winged (dipteran) flies, there aregiant chromosomes, called polytene chromosomes, that contain about 1000 DNA molecules laterally aligned.Each of these chromosomes has a length and cross-sectional diameter many times greater than those of thecorresponding chromosome at mitotic metaphase in ordinary somatic cells, as well as a constant and distinctivepattern of transverse banding (Figure 6.13).
The polytene structures are formed by repeated replication of the DNAin a closely synapsed pair of homologous chromosomes without separation of the replicated chromatin strands orof the two chromosomes. Polytene chromosomes are atypical chromosomes and are formed in "terminal" cells; thatis, the larval cells containing them do not divide and are eliminated in the formation of the pupa. Although they donot contribute to the tissues in the adult fly, the polytene tissues of larvae have been especially valuable in thegenetics of Drosophila, as will become apparent in Chapter 7.In polytene nuclei of D.
melanogaster and other dipteran species, large blocks of heterochromatin adjacent to thecentromeres are aggregated into a single compact mass called the chromocenter. Because the two largestchromosomes in Drosophila (chromosomes numbered 2 and 3) have centrally located centromeres, thechromosomes appear in the configuration shown in Figure 6.14: The paired X chromosomes (in a female), the leftand right arms of chromosomes 2 and 3, and a short chromosome (chromosome 4) project from the chromocenter.In a male, the Y chromosome, which consists almost entirely of heterochromatin, is incorporated in thechromocenter.The darkly staining transverse bands in polytene chromosomes have about a tenfold range in width. These bandsresult from the side-by-side alignment of tightly folded regions of the individual chromatin strands that are oftenvisible in mitotic and meiotic prophase chromosomes as chromomeres.
More DNA is present within the bands thanin the interband (lightly stained) regions. About 5000 bands have been identified in the D. melanogaster polytenechromosomes. This linear array of bands, which has a pattern that is constant and characteristic for each species,provides a finely detailed cytological map of the chromosomes. The banding pattern is such that observers withsufficient training and experience can identify short regions in any of the chromosomes (Figure 6.14).Because of their large size and finely detailed morphology, polytene chromosomes are exceedingly useful for aprocess called in situ nucleic acid hybridization.
In the procedure of in situ hybridization, nucleiFigure 6.13The polytene fourth chromosome of Drosophila melanogasteradhering to the chromocenter (positioned to the left, notshown). The somatic chromosomes of Drosophila, drawn toscale with respect to the polytene fourth chromosome,are shownat the upper right as they appear inmitotic prophase.[From C. Bridges. 1935. J. Heredity, 26: 60.]Page 235Figure 6.14Polytene chromosomes from a larval salivarygland cell of Drosophila melanogaster.
Thechromocenter is the central region in whichthe centromeric regions of all chromosomes areunited.[Courtesy of George Lefevre.]containing polytene chromosomes are squashed and the chromosomal DNA denatured, after which labeled probeDNA or RNA is added under conditions that favor renaturation. After washing, the only probe that remains in thechromosomes has formed hybrid duplexes with chromosomal DNA, and its position can be identified cytologically(Figure 6.15).Figure 6.15Autoradiogram of Drosophila melanogasterpolytene chromosomes hybridized insitu with radioactively labeled RNAcopied from the histone genes, showinghybridization to a particular region (arrow).This region identifies the position of thehistone genes in the chromosomes.[Courtesy of Mary Lou Pardue.]6.6—Repetitive Nucleotide Sequences in Eukaryotic GenomesIn bacteria, the variation in average base composition from one part of the genome to another is quite small.However, in eukaryotes, some components of the genome can be detected because their base composition is quitedifferent from the average of the rest of the genome.
For example, one component of crab DNA is only 3 percent G+ C, compared to an average of approximately 50 percent G + C, in the rest of the genome. The components withunusually low or unusually high G + C contents are called satellite DNA. In the mouse, satellite DNA accounts forabout 10 percent of the genome.
A striking feature of satellite DNA is that it consists of fairly short nucleotidesequences that may be repeated as many as a million times in a haploid genome. Other repetitive sequences alsoare present in eukaryotic DNA. Because repetitive DNA consists of many highly similar or identical sequences,fragments of repetitive DNA renature more readily than fragments of nonrepetitive DNA. Information about thesize of repeated sequences and the number of copies of a particular sequence can be obtained through studies of therate of renaturation. The quantitative analysis of DNA renaturation is considered next.Page 236Kinetics of DNA RenaturationAs we saw in Chapter 5, the rate-limiting step in the renaturation of separated DNA strands is the initial collisionbetween two complementary single strands. Because the chance of initial collision is concentration-dependent, therate of reassociation increases with DNA concentration (Figure 6.16A).
Any increase in DNA concentration resultsin a corresponding increase in the number of potential pairing partners for a given strand. The study of DNArenaturation has contributed greatly to our understanding of the number and types of DNA sequences present invarious genomes.To illustrate the analysis of reassociation, we begin by comparing the renaturation rates of solutions of DNAmolecules from the bacteriophages T7 and T4, which have no common base sequences and different molecularweights, and the T7 genome is smaller than that of T4.
In solutions in which the number of grams of DNA permilliliter is the same, the molar concentration of T7 is greater than that of T4. Thus if each solution is separatelydenatured and renatured, the molecules of T7 will renature more rapidly than those of T4. If the two solutions areinstead mixed, the T7 and T4 will renature independently of one another (because they are not homologous), and acurve such as that in Figure 6.16B will be obtained. Note that the curve consists of two steps, one for the morerapidly renaturing T7 molecules and the other for the T4 molecules. Each step in the curve accounts for half of thechange in the absorption of the solution at 260 nanometers (A260) because the initial concentration (in µg ofDNA/ml, which is proportional to A260) of each type of molecule was the same.As is apparent in Figure 6.16A, the renaturation curve of a molecule that contains no repeating base sequencesconsists of only a single step, and the rate of renaturation is a function of the size of the molecule.
If suchmolecules are fragmented into many components of equal size, then the molar concentration of each componentwill be the same as that of the unbrokenFigure 6.16(A) Dependence of renaturation time on the concentration of phage T7 DNA. After a period at 90°C to separate thestrands, the DNA was cooled to 60°C. Renaturation is complete when the relative absorption reaches 1. (B)Renaturation of a mixture of T4 and T7 DNA, each at the same temperature. Extrapolation (black dashedline) yields the early portion of the T4 curve; the ratio of the absorptions at points x and y yields the fractionof the total DNA that is T4 DNA.
The times required for half-completion of renaturation, t1/2, are obtained bydrawing the red horizontal lines, which divide each curve equally in the vertical direction, and then extendingthe red vertical lines to the time axis.Page 237molecule, so the renaturation rate should be unchanged by fragmentation. The rate actually decreases somewhat,but the important point is that if a DNA molecule contains only a single nonrepetitive sequence of base pairs, thenbreakage of the molecules does not yeild a renaturation curve consisting of steps. In contrast, if a molecule with anoverall sequence that includes both a unique component and several copies of different repeated sequences isfragmented, fragments containing the more numerous repeated sequences will renature more rapidly than thefragments containg portions of the unique sequence. For example, consider a molecule containing 50,000 basepairs and consisting of 100 copies of a tandemly repeated sequence of 500 base pairs.
If the molecules are brokeninto about 100 fragments of roughly equal size, each fragment will be about 500 base pairs. Althugh therenaturation curve for the fragments will have a single step, the renaturation rate will be characteristic of molecules500 nucleotides in length.
If a genome contains multiple families of repeated sequences whose abundances differ,the renaturation curve will have steps—one step for each repeating sequence. This principle is the basis of theanalysis of renaturation kinetics.Renaturation kinetics can be described in a simple mathematical form, because the reaction is one in which theratelimiting step is the initial collision of two molecules. In such a case, the fraction of single strands remainingdenatured at a time t after the start of renaturation is given by the expressionin which C is the concentration of single-stranded DNA in moles of nucleotide per liter, C0 is the initialconcentration, and k is a constant.
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