1 MCB 502A - 2015. Lecture #4 Genomes VS Chromosomes. DNA degradation. Differential DNA strand labeling as a detection approach. SCE. Besides confirming the semiconservative mode of DNA replication, Taylor’s results illuminated a new phenomenon. Occasionally, after the 2nd replication round, some radioactivity would be found in the otherwise non-radioactive chromosome. Remarkably, in all these cases, the otherwise radioactive chromosome of the bivalent would be always non-radioactive in the corresponding segment! This observation suggested a reciprocal swapping of chromosomal arms, subsequently called “sister-chromatid exchange” (SCE). Due to the low density of radioactive labeling, the original observation of Taylor was not highly convincing. However, sister-chromatid exchange in cells of higher eukaryotes (higher plants, mammals) was eventually fully confirmed by chemical methods of chromosomal staining, in particular by the method employing bromodeoxyuridine (BrdU), an analog of thymidine. Cells are grown in the presence of BrdU for two generations, the second anaphase is blocked, the chromosomes are spread and then stained with BrdU-specific dyes. This staining allows good distinction between the chromatid in the bivalent in which only one DNA strand is BrdU-labeled and its sister chromatid in which both DNA strands are BrdU-labeled. Sister-chromatid exchanges are seen as reciprocal changes in the labeling pattern between the two sister chromosomes, still attached to each other at the centromere. We now know that sister-chromatid exchanges indicate instances of recombinational repair of double-strand DNA breaks (more on it later). Multiple exchanges in certain mutants produce the pattern called “harlequin chromosomes”, characteristic of defects in the DNA metabolism, associated with some cancer-predisposition syndromes in humans. Can one apply this spectacular differential labeling to detect sister-chromatid exchange in prokaryotes? Not directly, because 1) prokaryotic chromosomes are small and cannot be seen in detail under the light microscope; 2) prokaryotic chromosomes are not condensed into compact bodies at any part of the cell cycle, unlike eukaryotic chromosomes at the metaphase of mitosis. Therefore, direct visualization is not available in prokaryotes. But detection of the DNA strand exchange by differential strand labeling is possible in bacteria, if combined with other methods. For example, Steiner and Kuempel suspected a site-specific recombination-catalyzed strand exchange in a particular region of the E. coli chromosome. To detect this exchange, they grew cells in a heavy medium, supplemented with 15N and 13C, and then switched them to a light, 14N 12C-containing medium for one generation. DNA duplexes became half-light, half-heavy. The researchers sheared the DNA to a relatively low MW, separated the two strands of the hybrid duplexes in alkaline (DNA denaturing) density gradient and observed the two expected peaks, corresponding to the heavy and the light DNA strands. They collected fractions covering these peaks, spotted them on a nylon membrane and hybridized with a probe centered on the small DNA region, in which they suspected a frequent site-specific strand exchange. Such an exchange would generate strands, in which heavy DNA is joined with light DNA at the point of exchange. The Dot-hybridization with a region-specific probe indeed revealed three peaks instead of two! Now there was an additional peak of intermediate density, exactly what was expected if there were a strand exchange in this sequence. As a negative control, the dot-hybridization with probes away from the region revealed only two peaks, the heavy one and the light one. These are powerful experimental approaches to detect strand exchange, which exploit the fact that DNA replicates semiconservatively.2 "One Picture is Worth a Thousand Words." Truism The picture of the replicating chromosome (Cairns) You have probably noticed, that the demonstration of semiconservative replication did not resolve the issue of DNA duplex rotation during replication, — it just confirmed that bacterial cells are capable of unwinding over 450,000 turns in 45 minutes (10,000 revolutions per minute!). It seemed impossible that a single replication point could rotate with such a speed. Therefore, it was proposed that the actual number of replication points is significant, so this rotation is spread among many points going concurrently. For example, if there were 100 replication points in the E. coli chromosome, the speed of DNA rotation during replication at any single point would be only 100 revolutions per minute — quite a different story. At that time nothing was known not only about how the E. coli chromosome replicates, but also about how the genomic DNA of E. coli is organized into its chromosome. Genetic data on marker transfer suggested a single circular chromosome of an enormous size, whereas physical measurements of the DNA pieces extracted from cells of any organism showed that DNA is of the uniform size of about 5-10 kbp. (Only later it was found that this uniform size is produced by shearing in syringes, used to distribute and manipulate DNA samples before the modern pipettors arrived.) If the chromosomes were really represented by a collection of short linear pieces, the circularity of genetic maps could have been due to the overlapping nature of these small DNA segments. John Cairns decided to see directly the real shape of the E. coli chromosomal DNA and how it replicates. Since it was suspected that long DNA molecules are extremely fragile, he absolutely minimized manipulations that could have been contributing to chromosomal breakage. He labeled the DNA of live cells with 3H-thymine, put the cells in a dialysis chamber and then slowly lysed them by changing the buffer in the adjacent chamber, separated from the first one by a semi-permeable membrane. He then allowed the contents of the lysed cells to slowly adsorb to a filter paper, dried the paper, exposed it to an autoradiography film and examined the images under the light microscope. He has seen a lot of incompletely freed bundles of DNA, he has detected many big structures suggesting that 1) E. coli DNA is a single molecule; 2) it is quite long. Eventually, Cairns started seeing huge and fully circular molecules and even molecules with internal loops, but unfortunately only broken ones. Finally, Cairns
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