MCB 502A - 2015. Lecture #4!Genomes vs Chromosomes. DNA degradation. — Differential DNA strand labeling as a detection approach. SCE. — The picture of the replicating chromosome (Cairns)Chromosomal organization of genomic DNA— Genome functions— The chromosomal organization of the genome— The differences between prokaryotic and eukaryotic chromosomesDNA degradation— The basic configurations of the natural DNA molecules— Gel electrophoresis— Southern blotting (detection of DNA degradation in vivo)— TCA precipitation— Major nucleases of E. coliDifferential DNA strand labeling as a detection approach— What is wrong with this scheme?The bivalent was replicated conservatively!Sister-chromatid exchange (SCE)-1— Besides establishing …, Taylor’s results illuminated …. — Occasionally, after the 2nd replication round, …. — Remarkably, in all these cases, the otherwise radioactive chromosome… — This observation suggested a reciprocal swapping of chromosomal arms, … (SCE). SCESister-chromatid exchange (SCE)-2— Due to the low density of radioactive labeling, … — However, sister-chromatid exchange <…> by chemical methods of chromosomal staining, … (BrdU) — Protocol: — cells are grown in the presence of BrdU for two generations;— the second anaphase is blocked— the chromosomes are spread and stained with BrdU-specific dyes. — … distinction between … one BrdU-labeled strand … both DNA strands are BrdU-labeled.Sister-chromatid staining with BrdU— Sister-chromatid exchanges … reciprocal changes … between the two sister chromosomes…. — … indicate instances of recombinational repair …. J. Bodycote and S. WolffSister-chromatid exchange (SCE)-3— Multiple exchanges … “harlequin chromosomes”, … defects in the DNA metabolism… J. Bodycote and S. WolffcontrolSister-chromatid exchange (SCE)-4Normal cells Bloom's syndrome cells Normal cells treated with a DNA-damaging agent MMS http://www.pnas.org/content/98/15/8196/F1.expansion.htmlSister-chromatid exchange (SCE)-5— …applicable to prokaryotes? — … 1) prokaryotic chromosomes are small…; 2) unlike eukaryotic sister chromatids … , prokaryotic … are never condensed … at any part of the cell cycle. — … direct visualization is not available …. — However, detection of the DNA strand exchange by differential strand labeling ….Sister-chromatid exchange (SCE)-6— For example, Walter Steiner and Peter Kuempel suspected a site-specific recombination-catalyzed strand exchange in a particular region of the E. coli chromosome. Frank Stahl Walter SteinerThis is not Peter Kuempel!Sister-chromatid exchange (SCE)-6— To detect this exchange, they grew cells in a heavy medium … and then switched them to a light … medium for one generation. — DNA duplexes became …— The researchers sheared the DNA … separated the two strands … in alkaline density gradient and observed … x x difhttp://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1998.00651.x/full+Sister-chromatid exchange (SCE)-7— They collected fractions spanning both peaks, spotted … and hybridized with … (dif), …. site-specific strand exchange. — Such an exchange would generate …. — The Dot-hybridization … — Now there was an additional peak …. — As a negative control, … http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1998.00651.x/fullSister-chromatid exchange (SCE)-8— These are powerful experimental approaches to detect strand exchange, which exploit the fact that DNA replicates semiconservatively.The picture of the replicating chromosome (Cairns)-1— … the demonstration of semiconservative replication did not resolve the issue of DNA duplex rotation …— it just confirmed that bacterial cells are capable of unwinding over 450,000 turns in 45 minutes… — It seemed impossible that a replication point could rotate …. — …is the actual number of replication points significant?. — … 100 replication points in the E. coli chromosome … only 100 revolutions per minute …The picture of the replicating chromosome (Cairns)-2— At that time nothing was known … E. coli chromosome replicates … organized …. — Genetic data … a single circular chromosome … physical measurements of the extracted DNA … uniform size of about 5-10 kbp. — (later: … shearing in syringes….) — … if indeed small, … circular genetic maps could have been...The picture of the replicating chromosome (Cairns)-3— John Cairns decided to find out…— … absolutely minimized manipulations … chromosomal breakage. — He labeled the DNA of live cells with 3H-thymine, …slowly lysed…. — … slowly adsorbed to a filter paper….cellsLysis bufferLysis bufferThe picture of the replicating chromosome (Cairns)-4— He has seen … bundles of DNA, … big structures suggesting that: 1) E. coli DNA is a single molecule; 2) it is incredibly long, as if all genome of E. coli is packed in a single DNA molecule. — Eventually, Cairns started seeing huge and fully circular molecules …The picture of the replicating chromosome (Cairns)-5— Finally, Cairns found a single picture …. — It apparently captured …. — Cairns measured … — … it was a single picture, so no hard conclusions …Single-picture results are like…The picture of the replicating chromosome (Cairns)-6— Taken at face value, …: 1) E. coli has a single circular DNA molecule; 2) this DNA molecule is replicated…. — In principle, theta-replication … in plasmids … whose length is … 1/100-1/1,000 of the chromosomal length. Replicating lambda DNA (Ross Inmann)Theta-replicating plasmidshttp://nar.oxfordjournals.org/content/30/3/656/F3.expansionThe picture of the replicating chromosome (Cairns)-6— … Gerry Wake’s pictures … for Bacillus subtilis.http://www.sciencedirect.com/science/article/pii/0022283673902234#The picture of the replicating chromosome (Cairns)-7Thus, the worst-case scenario turned out to be true for bacteria: — DNA is replicated semiconservatively. — There is a single, circular chromosome. — There are only one or two growing points on this chromosome.The picture of the replicating chromosome (Cairns)-8The problem of unwinding and rewinding of this DNA structure must be daunting. John CairnsSanta Maria de ColomboMultiple replication bubbles in the eukaryotic chromosomes— … the idea of multiple
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