scholarly journals Replication dynamics of individual loci in single living cells reveal variation of stochasticity

2018 ◽  
Author(s):  
Bénédicte Duriez ◽  
Sabarinadh Chilaka ◽  
Jean-François Bercher ◽  
Eslande Hercul ◽  
Nicole Boggetto ◽  
...  
Keyword(s):  
Replication Timing ◽  
S Phase ◽  
Timing Control ◽  
Homologous Chromosomes ◽  
Stochastic Nature ◽  
Time Period ◽  
Single Living Cells ◽  
Phase Out ◽  
Single Living ◽  
Eukaryotic Genomes

AbstractEukaryotic genomes are replicated under the control of a highly sophisticated program during the restricted time period corresponding to S-phase. The most widely used replication timing assays, which are performed on populations of millions of cells, suggest that most of the genome is synchronously replicated on homologous chromosomes. We investigated the stochastic nature of this temporal program, by comparing the precise replication times of allelic loci within single vertebrate cells progressing through S-phase at six loci replicated from very early to very late. We show that replication timing is strictly controlled for the three loci replicated in the first half of S-phase. Out of the three loci replicated in the second part of S-phase, two present a significantly more stochastic pattern. Surprisingly, we find that the locus replicated at the very end of S-phase, presents stochasticity similar to those replicated in early S-phase. We suggest that the richness of loci in efficient origins of replication, which decreases from early-to late-replicating regions, may underlie the variation of timing control during S-phase.

scholarly journals Replication timing control can be maintained in extrachromosomally amplified genes.

1991 ◽  
Vol 11 (9) ◽  
pp. 4779-4785 ◽  
Author(s):  
S M Carroll ◽  
J Trotter ◽  
G M Wahl
Keyword(s):  
Adenosine Deaminase ◽  
Timing Analysis ◽  
Single Gene ◽  
Replication Timing ◽  
Virus Genome ◽  
S Phase ◽  
Timing Control ◽  
Barr Virus ◽  
Extrachromosomal Elements

Extrachromosomal elements are common early intermediates of gene amplification in vivo and in cell culture. The time at which several extrachromosomal elements replicate was compared with that of the corresponding amplified or unamplified chromosomal sequences. The replication timing analysis employed a retroactive synchrony method in which fluorescence-activated cell sorting was used to obtain cells at different stages of the cell cycle. Extrachromosomally amplified Syrian hamster CAD genes (CAD is an acronym for the single gene which encodes the trifunctional protein which catalyzes the first three steps of uridine biosynthesis) replicated in a narrow window of early S-phase which was approximately the same as that of chromosomally amplified CAD genes. Similarly, extrachromosomally amplified mouse adenosine deaminase genes replicated at a discrete time in early S-phase which approximated the replication time of the unamplified adenosine deaminase gene. In contrast, the multicopy extrachromosomal Epstein-Barr virus genome replicated within a narrow window in late S-phase in latently infected human Rajii cells. The data indicate that localization within a chromosome is not required for the maintenance of replication timing control.

scholarly journals Replication dynamics of individual loci in single living cells reveal changes in the degree of replication stochasticity through S phase

2019 ◽  
Vol 47 (10) ◽  
pp. 5155-5169 ◽  
Author(s):  
Bénédicte Duriez ◽  
Sabarinadh Chilaka ◽  
Jean-François Bercher ◽  
Eslande Hercul ◽  
Marie-Noëlle Prioleau
Keyword(s):  
Living Cells ◽  
S Phase ◽  
Single Living Cells ◽  
Single Living

scholarly journals Replication timing control can be maintained in extrachromosomally amplified genes

1991 ◽  
Vol 11 (9) ◽  
pp. 4779-4785
Author(s):  
S M Carroll ◽  
J Trotter ◽  
G M Wahl
Keyword(s):  
Adenosine Deaminase ◽  
Timing Analysis ◽  
Single Gene ◽  
Replication Timing ◽  
Virus Genome ◽  
S Phase ◽  
Timing Control ◽  
Barr Virus ◽  
Extrachromosomal Elements

Extrachromosomal elements are common early intermediates of gene amplification in vivo and in cell culture. The time at which several extrachromosomal elements replicate was compared with that of the corresponding amplified or unamplified chromosomal sequences. The replication timing analysis employed a retroactive synchrony method in which fluorescence-activated cell sorting was used to obtain cells at different stages of the cell cycle. Extrachromosomally amplified Syrian hamster CAD genes (CAD is an acronym for the single gene which encodes the trifunctional protein which catalyzes the first three steps of uridine biosynthesis) replicated in a narrow window of early S-phase which was approximately the same as that of chromosomally amplified CAD genes. Similarly, extrachromosomally amplified mouse adenosine deaminase genes replicated at a discrete time in early S-phase which approximated the replication time of the unamplified adenosine deaminase gene. In contrast, the multicopy extrachromosomal Epstein-Barr virus genome replicated within a narrow window in late S-phase in latently infected human Rajii cells. The data indicate that localization within a chromosome is not required for the maintenance of replication timing control.

scholarly journals Two alleles of a developmentally regulated alpha-tubulin locus in Physarum polycephalum replicate on different schedules.

1993 ◽  
Vol 13 (1) ◽  
pp. 449-461 ◽  
Author(s):  
D B Cunningham ◽  
W F Dove
Keyword(s):  
Replication Timing ◽  
S Phase ◽  
Temporal Organization ◽  
Developmentally Regulated ◽  
Alpha Tubulin ◽  
Homologous Chromosomes ◽  
Allele Specific ◽  
Rna Blots ◽  
Temporal Windows ◽  
Mitotic Synchrony

The replication timing of a pair of natural alleles was compared at two alpha-tubulin loci of the Physarum plasmodium. Taking advantage of the naturally synchronous cell cycle of nuclei within the syncytial plasmodium, we analyzed the replication schedule of specific DNA fragments to a resolution of 10-min intervals within a 3-h S phase. At this level of resolution, differences in replication timing between polymorphic alleles at the same locus can be detected in a heterozygote. Specifically, the 3' region of the altA1 allele completes replication at between 20 and 40 min of S phase. The same region of the altA2 allele completes replication at between 40 and 80 min of S phase. In contrast, both alleles at the altB locus replicate concurrently within the first 10 to 15 min of S phase. Previous studies showed that both altA and altB are expressed in the plasmodium, their message levels peaking at mitosis, just minutes before the onset of S phase. However, altB message is detected at substantially higher levels than altA message on Northern (RNA) blots. The temporal windows over which the altA alleles each replicate are very broad in comparison with the levels of mitotic synchrony and altB replication synchrony in a single plasmodium. The allele-specific replication schedule of the altA locus demonstrates that the temporal organization of replicons is not strictly conserved between homologous chromosomes.

Two alleles of a developmentally regulated alpha-tubulin locus in Physarum polycephalum replicate on different schedules

1993 ◽  
Vol 13 (1) ◽  
pp. 449-461
Author(s):  
D B Cunningham ◽  
W F Dove
Keyword(s):  
Replication Timing ◽  
S Phase ◽  
Temporal Organization ◽  
Developmentally Regulated ◽  
Alpha Tubulin ◽  
Homologous Chromosomes ◽  
Allele Specific ◽  
Rna Blots ◽  
Temporal Windows ◽  
Mitotic Synchrony

The replication timing of a pair of natural alleles was compared at two alpha-tubulin loci of the Physarum plasmodium. Taking advantage of the naturally synchronous cell cycle of nuclei within the syncytial plasmodium, we analyzed the replication schedule of specific DNA fragments to a resolution of 10-min intervals within a 3-h S phase. At this level of resolution, differences in replication timing between polymorphic alleles at the same locus can be detected in a heterozygote. Specifically, the 3' region of the altA1 allele completes replication at between 20 and 40 min of S phase. The same region of the altA2 allele completes replication at between 40 and 80 min of S phase. In contrast, both alleles at the altB locus replicate concurrently within the first 10 to 15 min of S phase. Previous studies showed that both altA and altB are expressed in the plasmodium, their message levels peaking at mitosis, just minutes before the onset of S phase. However, altB message is detected at substantially higher levels than altA message on Northern (RNA) blots. The temporal windows over which the altA alleles each replicate are very broad in comparison with the levels of mitotic synchrony and altB replication synchrony in a single plasmodium. The allele-specific replication schedule of the altA locus demonstrates that the temporal organization of replicons is not strictly conserved between homologous chromosomes.

scholarly journals Development of Raman Imaging System for time-course imaging of single living cells

2010 ◽  
Vol 24 (1-2) ◽  
pp. 131-136 ◽  
Author(s):  
A. Zoladek ◽  
F. Pascut ◽  
P. Patel ◽  
I. Notingher
Keyword(s):  
Time Course ◽  
Imaging System ◽  
Living Cells ◽  
Spectral Measurements ◽  
Time Period ◽  
Single Living Cells ◽  
Raman Spectral ◽  
Single Living ◽  
Extended Time Period

Development of novel inverted Raman micro-spectrometer with the ability to perform multi-hours spectral measurements on living cells is presented. Our system combines a Confocal Raman Micro-Spectrometer and Fluorescence Microscope with cell incubator enclosure allowing measurement of cells in extended time period. To illustrate the feasibility of this Raman micro-spectroscopy system forin vitrotime-course studies of cells we performed an experiment where the same group of cells were scanned with the laser at 2 hours intervals between the scans over 8 hours to build Raman spectral images and ensure that no changes occur due to laser damage or environmental conditions. Cell viability test was performed with fluorescence microscopy on exactly the same cells at the end of the time-course Raman measurements.

Application of FRAP and digitized fluorescence microscopy to problems in cell membrane dynamics

1988 ◽  
Vol 46 ◽  
pp. 118-119
Author(s):  
K. Jacobson ◽  
A. Ishihara ◽  
B. Holifield ◽  
F. Zhang
Keyword(s):  
Fluorescence Microscopy ◽  
Cell Biology ◽  
Extended Period ◽  
Dynamic Structure ◽  
Living Cells ◽  
Membrane Dynamics ◽  
Molecular Cell Biology ◽  
Image Averaging ◽  
Single Living Cells ◽  
Single Living

Our laboratory is concerned with understanding the dynamic structure of the plasma membrane with particular reference to the movement of membrane constituents during cell locomotion. In addition to the standard tools of molecular cell biology, we employ both fluorescence recovery after photo- bleaching (FRAP) and digitized fluorescence microscopy (DFM) to investigate individual cells. FRAP allows the measurement of translational mobility of membrane and cytoplasmic molecules in small regions of single, living cells. DFM is really a new form of light microscopy in that the distribution of individual classes of ions, molecules, and macromolecules can be followed in single, living cells. By employing fluorescent antibodies to defined antigens or fluorescent analogs of cellular constituents as well as ultrasensitive, electronic image detectors and video image averaging to improve signal to noise, fluorescent images of living cells can be acquired over an extended period without significant fading and loss of cell viability.

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