Recollections of a Helmstetter Disciple
<p>Scheme for construction of minichromosomes. Red color denotes plasmid DNA. Chromosomes are shown in a fast growth configuration used to increase the relative copies of <span class="html-italic">oriC</span>. See text for a description of the figure.</p> "> Figure 2
<p><span class="html-italic">OriC</span> cloning reports from the weekly lab newsletter “The Flash”. See text.</p> "> Figure 3
<p>Baby machine analysis of minichromosome cell cycle replication. (<b>A</b>). Diagram of baby machine apparatus and sample collection. Cells growing exponentially were labeled with [<sup>3</sup>H]thymidine for 4 min, bound to a membrane filter, and eluted with glucose/Casamino acids minimal medium. (<b>B</b>). Electrophoretic separation of labeled chromosome and pAL49 minichromosome DNA from new daughter cells. Whole-cell lysates of new daughter cells in the effluent were subjected to agarose gel electrophoresis and fluorography. The radioactive bands corresponding to chromosomal and pAL49 DNA are shown for consecutive 4 min samples of the effluent. Exposure times to the x-ray films were 3 h for the chromosomal bands and 10 days for the minichromosome bands. Modified from reference [<a href="#B10-life-13-01114" class="html-bibr">10</a>].</p> "> Figure 4
<p>(Left panel) Timing of chromosome and pAL49 minichromosome replication during the division cycle. Exponential-phase cultures of <span class="html-italic">E. coli</span> B/r F26(pAL49) growing in glucose plus Casamino Acids (<b>a</b>), glucose plus six amino acids (<b>b</b>), glucose (<b>c</b>), or glycerol (<b>d</b>) were pulse-labeled and treated as described in the legend to <a href="#life-13-01114-f003" class="html-fig">Figure 3</a>. The radioactivity per cell in minichromosome DNA (closed circles) and total radioactivity per cell (open circles) in newborn cells collected from the effluents of membrane-bound cultures are plotted at the midpoints of the 4 min collection intervals. Abrupt increases in radiolabel (reading right to left) indicate the time of initiation of chromosomal DNA replication. (Right panel). Minichromosome replication during the division cycle of <span class="html-italic">E. coli</span> B/r F(pAL49) growing at different rates. Cells growing exponentially in minimal medium containing glucose plus Casamino acids (<b>a</b>), glucose plus six amino acids (<b>b</b>), or glucose alone (<b>c</b>) were pulse-labeled with [<sup>3</sup>H]thymidine for 4 min, bound to a membrane filter, and eluted with minimal medium of the same composition. Whole-cell lysates of the newborn cells were treated as in <a href="#life-13-01114-f003" class="html-fig">Figure 3</a>. Radioactivity corresponding to closed circular pAL49 minichromosome DNA is shown for consecutive 4 min samples of the effluent at each growth rate. The cell concentrations are also shown, and the vertical interrupted lines indicate the end of each generation of growth on the membrane. Modified from reference [<a href="#B11-life-13-01114" class="html-bibr">11</a>].</p> "> Figure 5
<p>Cell cycle replication of various plasmids. (<b>A</b>). Fluorograph of radioactive plasmid DNA in newborn cells from the effluent of a membrane filter-bound culture of <span class="html-italic">E. coli</span> B/rF26 containing pSG21 mini-F, pBR322, and pAL70 simultaneously. Cells were grown, pulse-labeled, and prepared as in <a href="#life-13-01114-f003" class="html-fig">Figure 3</a>. In this experiment, all lanes contained lysate from the same number of newborn cells. (<b>B</b>). Radioactive plasmid DNA in newborn cells from a membrane filter-bound culture of <span class="html-italic">E. coli</span> B/r F26 containing F’ lac, pSClO1, pAL49, and pBR322 simultaneously. Cells growing exponentially were pulse labeled and treated as described in <a href="#life-13-01114-f003" class="html-fig">Figure 3</a>. Modified from [<a href="#B16-life-13-01114" class="html-bibr">16</a>].</p> "> Figure 6
<p>(Left panel) Dye titrations of pAL2 and pBR322 closed circular DNA. pAL2 (<b>A</b>) and pBR322 (<b>B</b>) DNA was isolated from JTT1 recA grown at 37 °C and electrophoresed in gels containing increasing concentrations of ethidium bromide. pAL2 and pBR322 were electrophoresed through 0.6 and 0.8% agarose, respectively. The concentrations of EtBr (in hundredths of micrograms per milliliter) from left to right are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11. (I) and (II) are supercoiled and relaxed-nicked circular DNA, respectively. (Right panel) Positions of promoter sequences and the direction of transcription on minichromosomes pAL2, pAL20, pAL22, and pAL220 (indicated by arrows). Only pAL20 and pAL22 were able to replicate in <span class="html-italic">E. coli</span> strains (<span class="html-italic">topA, gyrB</span>) with decreased supercoiling. Modified from [<a href="#B27-life-13-01114" class="html-bibr">27</a>].</p> "> Figure 7
<p>Segregation of minichromosomes and chromosomes during baby machine analysis. Theoretical segregation patterns of radiolabeled DNA are shown for bound (on the horizontal line) and released cells growing with a generation time of C + D minutes. Four successive generations are shown. Old poles lacking attachment sites are shown by thick lines. (Upper panel) Minichromosome segregation is shown with cells that initially contain 20 copies. Average copy numbers of labeled molecules are shown to the left of each cell and the internal distribution of copies is shown above and below the dotted line representing the division septum. The radiolabeled copies in released cells are also shown. (Lower panel) Chromosome segregation. Radiolabeled chromosomal strands (assuming 2 chromosomes in the initially bound cell) are shown in a similar fashion to panel A. Modified from [<a href="#B37-life-13-01114" class="html-bibr">37</a>].</p> ">
Abstract
:1. Introduction
2. Making E. coli Minichromosomes and Cell Cycle Analysis
3. Searching for Cell Cycle-Specificity in Plasmid Systems
4. A Sidestep into the Role of DNA Supercoiling in Minichromosome Regulation
5. Studying the Non-Random Segregation of Minichromosomes
6. Discussion
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Leonard, A.C. Recollections of a Helmstetter Disciple. Life 2023, 13, 1114. https://doi.org/10.3390/life13051114
Leonard AC. Recollections of a Helmstetter Disciple. Life. 2023; 13(5):1114. https://doi.org/10.3390/life13051114
Chicago/Turabian StyleLeonard, Alan C. 2023. "Recollections of a Helmstetter Disciple" Life 13, no. 5: 1114. https://doi.org/10.3390/life13051114