MCB 201 Gene Expression - Spring Semester 2003
Lecture 10 (Organizing Cellular DNA into Chromosomes)
Section 9.5
Introduction: Most of our detailed understanding of the mechanism of transcription comes from in vitro studies. In test tube reactions (in vitro studies), the template for transcription is DNA to which is bound only a small fraction of the proteins that reside in chromatin in cells. How does cellular chromatin with its dense coat of proteins covering the DNA become a template for transcription inside the cell nucleus? To begin to answer this question, we need to consider chromosome structure, particularly the folding of DNA in chromosomes, and nuclear organization.
1. What can we learn from E. coli chromosome packaging that could help us understand the more complicated problem of folding eukaryotic chromatin? Figure 9-28. Electron micrograph of an isolated folded E. coli chromosome. The circular DNA molecule in the E. coli chromosome is about 1 mm long. It would occupy a volume about 1000 times the volume of an E. coli cell if simply coiled randomly. This large volume is mainly due to the charge repulsion of the negatively charged phosphate residues in the DNA backbone. Inside the cell, these charges are neutralized by small positively charged molecules called polyamines, e.g. spermine and spermidine. Also proteins such as H-NS bind to the DNA and help it to fold into a more compact structure. And finally, the DNA is tightly supercoiled, a twisted configuration like we discussed earlier for SV40 viral DNA (figure 4-11). E. coli uses the enzyme DNA gyrase to put these twists in the DNA, an ATP (energy) requiring process. In this electron micrograph, an isolated E. coli chromosome is shown which is highly supercoiled and attached to a fragment of cell membrane. Replication in E. coli is thought to occur in association with the plasma membrane.
2. Figure 9-29. Electron micrographs of chromatin extracted from eukaryotic cells in extended and condensed forms. The folding problem for eukaryotic DNA is much more complex simply because there is so much more DNA to fold. How eukaryotic chromatin appears after isolation depends heavily on the ionic strength of the buffer used to prepare it. Isotonic buffer has about the same salt concentration found in cells (~0.15M KCl, i.e. physiological ionic strength) and nuclei isolated at this concentration release chromatin that is about half protein and DNA by mass. It appears as a condensed 30 nm diameter fiber, as shown in panel B. Chromatin isolated in low ionic strength buffer has an unfolded or extended structure that looks similar to 'beads on a string'. The beads are nucleosomes of about 10 nm in diameter and the string is connecting DNA. This is thought to be the configuration of the chromatin during transcription.
3. Figure 9-30. Structure of the nucleosome. In eukaryotic cells, the problem of neutralizing the negative charge of the phosphodiester backbone of DNA is solved with histone proteins. These are basic proteins, rich in positively charged amino acids like Arg and Lys. Two molecules from each of four histone families assemble into a core (histones H2A, H2B, H3 and H4). About 146 base pairs or a little less than two turns of double stranded DNA are wound around a core. This DNA plus the histone core defines a nucleosome, which is the basic unit of packaging of eukaryotic chromatin. It is a structure that is highly concerved evolutionarily, and therefore must be fairly ancient.
4. Figure 9-31. Solenoid model of the 30-nm condensed chromatin fiber in a side view. There is a fifth histone family, called H1, which is bound to DNA on the inside of the solenoid in this model. Histone H1 is thought to be involved in either forming or stabilizing the solenoid structure. How does the chromatin decondense in vivo to allow transcription to occur? There is evidence now that acetylation of histone N-termini on lysine residues in this region of histones reduces chromatin condensation. The acetylation would reduce the positive charge of the histones in this domain, which is thought to interact with DNA phosphates. So histone acetylases and deacetylases are now the focus of intense study as major players in the regulation of gene expression by providing or restricting access to genes through effects on chromatin folding.
5. Figure 9-32. Demonstration that transcriptionally active genes are more susceptible than inactive genes to DNase I digestion. The extent of histone acetylation correlates with the sensitivity of chromatin DNA to digestion by nucleases. In the experiment summarized here, chick embryo erythroblasts actively synthesize globin, their histones have more acetylation, and the globin gene is more sensitive to digestion by DNase I. In contrast, cultured undifferentiated MSB cells do not synthesize globin, the globin gene is resistant to digestion by DNase I, and the histones in this region of the chromosome have low levels of acetylation. The method shown here is called Southern blotting. This is done by extracting nuclear DNA and cutting it with the restriction endonuclease BamH1 in this specific example. The DNA fragments were then separated by size in gels. The DNA in the gels was transfered to a filter and this filter was then incubated with a radioactive DNA probe containing globin gene sequence. This probe hybrizes on the filter to 4.6 Kb BamH1 fragments containing the globin gene.
Section 9.6 (Morphology and Functional Elements of Eukaryotic Chromosomes)
6. Figure 9-33. Karyotypes of the Reeves muntjac and the Indian muntjac, two species of small deer that are quite similar but do not interbreed. A karyotype consists of all of the metaphase chromosomes of a cell arranged, usually now by computer analysis, into homologous pairs in decreasing length with the pair of sex chromosomes placed at the end of the diagram. The karyotypes of two very similar species can be radically different. That is, the organization of genomes into individual chromosomes does not seem to follow much logical order, certainly not phylogenic relationships. However, chromosome number, size and morphology (i.e. karyotype) as seen at metaphase of the cell cycle are specifies-specific.
7. Figure 9-34. Electron micrograph of a histone-depleted metaphase chromosome prepared from HeLa cells by treatment with a mild detergent. A nonhistone protein scaffolding (dark electron-dense structure) is visible from which long loops of DNA extend, probably in the configuration of 30 nm fibers. The current thinking is that each of these loops can function independently of each other during gene expression. So, it should be possible for cells to unfold all or a portion of one of these loops to a beads-on-a-string configuration, essentially making genes in this loop accessible to the transcriptional machinery.
8. Figure 9-35. Model for the packing of chromatin and the chromosome scaffold in metaphase chromosomes. The diameters of the various configurations shown on the right side of the figure can be used to estimate the fold packing of the chromatin that occurs at each step.
9. Media Connection: Three-dimensional packing of nuclear chromosomes.
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