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MCB 502A-2015. Lecture #11. The Cell Cycle. — Helmstetter's "baby machine"— Replication period as a function of growth rate— The regulation of chromosomal initiation: titration of DnaA— The initiation cascade reflects the eclipse period— Flow cytometry reveals initiation synchrony— Dam and the origin sequestration— SeqA: the eclipse and beyond— Nucleoid administration Genome Logistics— DNA compactionHow to synchronize the culture without it noticing it?Baby machineHelmstetter's "baby machine"-1 — … synchronize …. without… synchronizing …? — … paradox, …. permanently stick…. — Instead …, Charles Helmstetter fractionated … age … pulse-labeling …Helmstetter's "baby machine"-2 — … label … culture… filtering … flushing … to remove the label. E. coli strain … attach permanently … — … inverted …. dripped …. without the label…. collecting the eluate …. Unlabeled cultureFilterFlipGrowth mediumAttach to another funnelCollect fractionsNewborn (labeled) cellsIndividual cells!Labeling for 1'The real setup— original … attached … the newly-born… membrane. — … 4-minute fractions… fractionated … age at … labeling.Helmstetter's "baby machine"-4 — … reasoning … : — if a bacterium was at the end of its cycle at the time of labeling, then its daughter cell would be eluted right away; — if bacterium was in the middle of its cell cycle, then its daughter would be eluted after half the cell cycle time; — whereas if the cell was just born at the time of labeling and attachment, its daughter cell would be eluted after a period of time equal to the division time under these growth conditions.Helmstetter's "baby machine"-5 — … expected number of cells… straight line? — … the theoretical elution curve … saw-tooth pattern: — … beginning … daughters of cells that were ready to divide before filtration, … end …. daughters of cells that were just born …. — … ratio of a cell … two daughters … 1:2. Elution time (generations)0 1 2 3 21Helmstetter's "baby machine"-6 — … growing culture … fission, newborn … twice as frequent … about to divide. — … plot . relative frequency of cells … age … line decreasing from 2 to 1… Elution time (generations)0 1 2 3 21Daughters of cells that were dividing at attachmentDaughters of cells that were newborn at attachmentHelmstetter's "baby machine"-7 — Helmstetter …. grew his E. coli in four …. carbon sources,…. 45, 60, 80 and 120 minutes:— … not going down to abscissa … newborn cells … stuck on the membrane, … overflow ….. — … possible … detaching with time from the membrane … .Helmstetter's "baby machine"-8 — … plot the rate of synthesis … function of the cell age. — If the macromolecule (A) … throughout … (… ribosome…), …rate of synthesis will increase…. — … pulse-label the macromolecule …. follow the radioactivity … observe a straight declining line …. — … semi-log … two-fold dilution …Helmstetter's "baby machine"-9 — … macromolecule … (B) … continuously… rate … doubles halfway … plot the step in relative synthesis rate … descending step function … function of the elution time. — … synthesized during the central 50% of the cell cycle,… haystack (starting from and returning to zero), … collection of two-fold-diminishing… .0 0.5 1Helmstetter's "baby machine"-10 — … Helmstetter observed … pulsed-labeled … four different media… eluted cells:— "Haystacks" in the acetate medium… … DNA synthesis … central 60 minutes …. — "Steps" in the glucose medium … doubles halfway … new initiation … termination ….Helmstetter's "baby machine"-11 — … elution patterns … measure in diverse…: 1) the generation time; 2) the length of the DNA synthesis period; 3) the relationship between the two periods. — … variety of conditions, … relatively non-invasive, … minimally perturbed.Various ways to synchronize without interfering with the cell cycle-1Concept-in-the-box. Synchronization without interference with growth of the culture is achieved by fractionation. — In general, the newly-born cells are two times smaller than the cells that are about to divide, and therefore can be separated from the latter by gradient centrifugation as, correspondingly, the slowest- and the fastest-sedimenting fractions. This approach works best for bacteria and yeast, and in general for organisms in which the hard cell envelope keeps the cell shape constant. — Cultured animal cells can be synchronized due to the fact that they become loosely attached to their substrate during mitosis. http://www.di.uq.edu.au/proj17aprochttp://www.sciencedirect.com/science/article/pii/0014482763903069#Various ways to synchronize without interfering with the cell cycle-2— … fractionation by time,… only the newly-born cells.— … the real power … combined … preceding pulse labeling. — Instead of experimenting … measured their radioactivity … parents … .— … later patented … synchronization … human lymphocytes… http://www.google.com/patents/US20020123144Limits of bacterial cell capacity to produce DNAReplication period as a function of growth rate-1— Helmstetter… others …— … pattern … other methods … complex and surprising: — … duration of the replication period … division time … bi-phasic. — … between 0.01 and 1 doublings per hour… . Division Replication % of divi-time (min) time (min) sion time1,200 200 1760 42 7024 42 175 (?)Replication period as a function of growth rate-3— … once … committed … poor medium… complete DNA synthesis…. — … a major commitment, … stockpile enough resources … …medium.Replication period as a function of growth rate-4— … bizarre… between 1 and 2.5 divisions per hour… 42 minutes … division rate … — … takes 70% of the division time … once every 60 minutes … takes 175% of the division time … once every 24 minutes … Division Replication % of divi-time (min) time (min) sion time1,200 200 1760 42 7024 42 175 (?)Replication period as a function of growth rate-5— …
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