scholarly journals An Artificially Constructed De Novo Human Chromosome Behaves Almost Identically to Its Natural Counterpart during Metaphase and Anaphase in Living Cells

2006 ◽  
Vol 26 (20) ◽  
pp. 7682-7695 ◽  
Author(s):  
Tomohiro Tsuduki ◽  
Megumi Nakano ◽  
Nao Yasuoka ◽  
Saeko Yamazaki ◽  
Teruaki Okada ◽  
...  
Keyword(s):  
Yeast Artificial Chromosome ◽  
Fluorescent Protein ◽  
De Novo ◽  
Artificial Chromosome ◽  
Time Lapse ◽  
Living Cells ◽  
Lac Repressor ◽  
Host Chromosome ◽  
Artificial Chromosomes ◽  
Spindle Midzone

ABSTRACT Human artificial chromosomes (HACs) are promising reagents for the analysis of chromosome function. While HACs are maintained stably, the segregation mechanisms of HACs have not been investigated in detail. To analyze HACs in living cells, we integrated 256 copies of the Lac operator into a precursor yeast artificial chromosome (YAC) containing α-satellite DNA and generated green fluorescent protein (GFP)-tagged HACs in HT1080 cells expressing a GFP-Lac repressor fusion protein. Time-lapse analyses of GFP-HACs and host centromeres in living mitotic cells indicated that the HAC was properly aligned at the spindle midzone and that sister chromatids of the HAC separated with the same timing as host chromosomes and moved to the spindle poles with mobility similar to that of the host centromeres. These results indicate that a HAC composed of a multimer of input α-satellite YACs retains most of the functions of the centromeres on natural chromosomes. The only difference between the HAC and the host chromosome was that the HAC oscillated more frequently, at higher velocity, across the spindle midzone during metaphase. However, this provides important evidence that an individual HAC has the capacity to maintain tensional balance in the pole-to-pole direction, thereby stabilizing its position around the spindle midzone.

Visualization of Membrane Sorting and Fusion in Living Cells using Total Internal Reflection (TIR) and Multicolor Video Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 34-35
Author(s):  
Derek Toomre ◽  
Patrick Keller ◽  
Elena Diaz ◽  
Jamie White ◽  
Kai Simons
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
Video Microscopy ◽  
Internal Reflection ◽  
Fast Time ◽  
Cellular Polarity ◽  
Microscopy Technique

Post-Golgi sorting of different classes of newly synthesized proteins and lipids is central to the generation and maintenance of cellular polarity. to directly visualize the dynamics and location of apical/basolateral sorting and trafficking we used fast time-lapse multicolor video microscopy in living cells. Specifically, green fluorescent protein color variants (cyan, CFP; yellow, YFP) of apical cargo (GPI-anchored) and basolateral cargo (vesicular stomatitis virus glycoprotein, VSVG) were generated; see FIG 1. Fast dual color fluorescence video microscopy allowed visualization with high temporal and spatial resolution. Our studies revealed that apical and basolateral cargo progressively segregated into large domains in Golgi/TGN structures, excluded resident proteins, and exited in separate transport containers. These carries remained distinct and did not merge with endocytic structures en route to the plasma membrane. Interestingly, our data suggest that the primary sorting occurs by lateral segregation in the Golgi, prior to budding (FIG 2). Further characterization of morphological differences of apical versus basolateral transport carriers was achieved using a specialized microscopy technique called total internal reflection (TIR) microscopy. with this approach only the bottom of the cell (<100 nm) was illuminated by an exponentially decaying evanescent “wave” of light. A series of images, taken at ∼1 second intervals, shows a bright “flash” of fluorescence when the vesicle fuse with the plasma membrane and the fluorophore diffuses into the plasma membrane (FIG 3).

scholarly journals Detection of myc translocations in lymphoma cells by fluorescence in situ hybridization with yeast artificial chromosomes

Blood ◽  
1995 ◽  
Vol 85 (8) ◽  
pp. 2132-2138 ◽  
Author(s):  
ML Veronese ◽  
M Ohta ◽  
J Finan ◽  
PC Nowell ◽  
CM Croce
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Yeast Artificial Chromosome ◽  
Human Lymphocytes ◽  
Artificial Chromosome ◽  
Chromosome 8 ◽  
Metaphase Chromosomes ◽  
Artificial Chromosomes ◽  
Myc Gene

Translocations involving chromosome 8 at band q24 and one of the Ig loci on chromosomes 14q32, 22q11, and 2p11 are the hallmark of Burkitt's lymphoma (BL). It has been previously observed that the exact localization of the breakpoints at chromosome 8q24 can vary significantly from patient to patient, scattering over a distance of more than 300 kb upstream of c-myc and about 300 kb downstream of c-myc. To generate probes for fluorescence in situ hybridization (FISH) that detect most c-myc translocations, we screened a yeast artificial chromosome (YAC) library from normal human lymphocytes by colony hybridization, using three markers surrounding the c-myc gene as probes. We obtained 10 YAC clones ranging in size between 500 and 200 kb. Two nonchimeric clones were used for FISH on several BL cell lines and patient samples with different breakpoints at 8q24. Our results show that the YAC clones detected translocations scattered along approximately 200 kb in both metaphase chromosomes and interphase nuclei. The sensitivity, rapidity, and feasibility in nondividing cells render FISH an important diagnostic tool. Furthermore, the use of large DNA fragments such as YACs greatly simplifies the detection of translocations with widely scattered breakpoints such as these seen in BL.

scholarly journals Germ-line transmission and developmental regulation of a 150-kb yeast artificial chromosome containing the human beta-globin locus in transgenic mice

1993 ◽  
Vol 90 (23) ◽  
pp. 11381-11385 ◽  
Author(s):  
K M Gaensler ◽  
M Kitamura ◽  
Y W Kan
Keyword(s):  
Transgenic Mice ◽  
Yeast Artificial Chromosome ◽  
Germ Line ◽  
Physiological Role ◽  
Artificial Chromosome ◽  
Chromatin Domain ◽  
Globin Genes ◽  
Beta Globin ◽  
Cis Acting ◽  
Artificial Chromosomes

Sequential expression of the genes of the human beta-globin locus requires the formation of an erythroid-specific chromatin domain spanning > 200 kb. Regulation of this gene family involves both local interactions with proximal cis-acting sequences and long-range interactions with control elements upstream of the locus. To make it possible to analyze the interactions of cis-acting sequences of the human beta-globin locus in their normal spatial and sequence context, we characterized two yeast artificial chromosomes (YACs) 150 and 230 kb in size, containing the entire beta-globin locus. We have now successfully integrated the 150-kb YAC into the germ line of transgenic mice as a single unrearranged fragment that includes the locus control region, structural genes, and 30 kb of 3' flanking sequences present in the native locus. Expression of the transgenic human beta-globin locus is tissue- and developmental stage-specific and closely follows the pattern of expression of the endogenous mouse beta-globin locus. By using homology-directed recombination in yeast and methods for the purification and transfer of YACs into transgenic mice, it will now be feasible to study the physiological role of cis-acting sequences in specifying an erythroid-specific chromatin domain and directing expression of beta-globin genes during ontogeny.

Arabidopsis YAC restriction mapping

Genome ◽  
1998 ◽  
Vol 41 (6) ◽  
pp. 806-817
Author(s):  
Shaun M Morroll ◽  
Zoe A Wilson
Keyword(s):  
Positional Cloning ◽  
Floral Development ◽  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
Genomic Region ◽  
Restriction Mapping ◽  
Artificial Chromosomes ◽  
Number Of Genes ◽  
Yeast Artificial Chromosomes ◽  
Large Genomic Region

The approach of partial restriction mapping and vector hybridisation has been used to restriction map and align six yeast artificial chromosomes (YACs) corresponding to the top arm (~27.9 centiMorgans, cM) of Arabidopsis chromosome 5 and confirm the chimeric nature of a further four clones which map to this region. The restriction endonucleases Sma1 and Sfi1 which recognise rare-medium frequency sites in the Arabidopsis genome were used. This work has restriction mapped a 315 kb region that includes a number of genes implicated in floral development, namely PISTILLATA and TOUSLED, and a number of uncharacterised genes involved in male gametogenesis (e.g., Ms1 and Ms37). The information generated can be used to transcriptionally map genes to this contig and will provide data for the isolation of several uncharacterised floral development genes which lie in this region. This approach has demonstrated how large tracts of YAC DNA can be mapped and aligned to show the presence/absence of chimeric YAC clones and provide detailed restriction knowledge for a large genomic region to help facilitate the positional cloning of genes.Key words: yeast artificial chromosome, YAC, Arabidopsis thaliana, partial restriction mapping, floral development.

scholarly journals Actin dynamics in living mammalian cells

1998 ◽  
Vol 111 (12) ◽  
pp. 1649-1658 ◽  
Author(s):  
C. Ballestrem ◽  
B. Wehrle-Haller ◽  
B.A. Imhof
Keyword(s):  
Actin Cytoskeleton ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Time Lapse ◽  
Living Cells ◽  
Actin Dynamics ◽  
Mammalian Cell Lines ◽  
Cytoskeletal Dynamics ◽  
Green Fluorescent

The actin cytoskeleton maintains the cellular architecture and mediates cell movements. To explore actin cytoskeletal dynamics, the enhanced green fluorescent protein (EGFP) was fused to human β-actin. The fusion protein was incorporated into actin fibers which became depolymerized upon cytochalasin B treatment. This functional EGFP-actin construct enabled observation of the actin cytoskeleton in living cells by time lapse fluorescence microscopy. Stable expression of the construct was obtained in mammalian cell lines of different tissue origins. In stationary cells, actin rich, ring-like structured ‘actin clouds’ were observed in addition to stress fibers. These ruffle-like structures were found to be involved in the reorganization of the actin cytoskeleton. In migratory cells, EGFP-actin was found in the advancing lamellipodium. Immobile actin spots developed in the lamellipodium and thin actin fibers formed parallel to the leading edge. Thus EGFP-actin expressed in living cells unveiled structures involved in the dynamics of the actin cytoskeleton.

A spinning disk confocal microscope system for rapid high resolution, multimode, fluorescence speckle microscopy and GFP imaging in living cells

2001 ◽  
Vol 7 (S2) ◽  
pp. 8-9
Author(s):  
Paul Maddox ◽  
Julie Canman ◽  
Sonia Grego ◽  
Wendy Salmon ◽  
Clare Waterman-Storer ◽  
...  
Keyword(s):  
High Resolution ◽  
Fluorescent Protein ◽  
Imaging System ◽  
Ccd Camera ◽  
High Sensitivity ◽  
Time Lapse ◽  
Living Cells ◽  
High Signal ◽  
High Quantum Efficiency ◽  
Spinning Disk

High resolution fluorescent speckle microscopy (FSM) and green fluorescent protein (GFP) imaging in living cells can require image recording at low densities of fluorophores (10 or less/resolvable unit) with low light excitation to prevent photobleaching. This needs efficient optical components, a high quantum efficiency detector, and a digital image acquisition and display system for time-lapse recording of multiple channels. Recently, Shinya and Ted Inoue have described the advantages of the Yokogawa CSU-10 spinning-disk confocal scanning unit for obtaining high quality fluorescent images with brief exposures and low fluorescence bleaching. Based on their findings, we have combined the CSU-10 unit with a high sensitivity pan-chromatic CCD camera to facilitate high spatial and temporal resolution imaging of fluorescence in living cells. in addition, the high signal-to-noise in images obtained with this instrument provides the opportunity to obtain 3-D views of extraordinary resolution and image quality after iterative constrained de-convolution.Our imaging system is constructed around a Nikon TE300 inverted microscope equipped with either a 60X or 100X Plan Apochromat objective, and standard epi-fluorescence optics for visual inspection of the specimen to locate cells for recording.

scholarly journals Connexin-specific distribution within gap junctions revealed in living cells

2000 ◽  
Vol 113 (22) ◽  
pp. 4109-4120 ◽  
Author(s):  
M.M. Falk
Keyword(s):  
High Resolution ◽  
Gap Junctions ◽  
Gap Junction ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Time Lapse ◽  
Living Cells ◽  
Dye Transfer ◽  
Channel Distribution ◽  
Specific Distribution

To study the organization of gap junctions in living cells, the connexin isotypes alpha(1)(Cx43), beta(1)(Cx32) and beta(2)(Cx26) were tagged with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. The cellular fate of the tagged connexins was followed by high-resolution fluorescence deconvolution microscopy and time-lapse imaging. Comprehensive analyses demonstrated that the tagged channels were functional as monitored by dye transfer, even under conditions where the channels were assembled solely from tagged connexins. High-resolution images revealed a detailed structural organization, and volume reconstructions provided a three-dimensional view of entire gap junction plaques. Specifically, deconvolved dual-color images of gap junction plaques assembled from CFP- and YFP-tagged connexins revealed that different connexin isotypes gathered within the same plaques. Connexins either codistributed homogeneously throughout the plaque, or each connexin isotype segregated into well-separated domains. The studies demonstrate that the mode of channel distribution strictly depends on the connexin isotypes. Based on previous studies on the synthesis and assembly of connexins I suggest that channel distribution is regulated by intrinsic connexin isotype specific signals.

scholarly journals Chromosome 11q23 translocations in both infant and adult acute leukemias are detected by in situ hybridization with a yeast artificial chromosome

Blood ◽  
1992 ◽  
Vol 80 (7) ◽  
pp. 1659-1665
Author(s):  
L Kearney ◽  
M Bower ◽  
B Gibbons ◽  
S Das ◽  
T Chaplin ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Yeast Artificial Chromosome ◽  
De Novo ◽  
Artificial Chromosome ◽  
Underlying Mechanism ◽  
Chromosome 11 ◽  
Acute Leukemias ◽  
Wide Range ◽  
Chromosome 11Q23

The yeast artificial chromosome (YAC-13HH4), which spans a 440-kb region of DNA just distal to the CD3 locus on chromosome 11 at band q23, has been used to characterize a range of chromosomal translocations in acute leukemias from both adults and infants. In situ hybridization was performed on metaphase cells from bone marrow of 17 leukemias and two cell lines with a variety of chromosome 11q23 abnormalities. It was established that in infant leukemias the translocations t(11;19), t(4;11), and t(5;11) had occurred in the region defined by YAC 13HH4. Additionally, the translocations t(4;11), t(6;11), t(9;11), t(X;11), and t(10;11) in other leukemias were found to disrupt the same region of chromosome 11q23, although an exception was found in one t(6;11) translocation for which the breakpoint was distal to the YAC. One patient had a t(9;11) translocation in a therapy- related leukemia, suggesting that this class of etoposide-related malignancy has similar breakpoints to those occurring in de novo leukemias. An example of a lymphoma-derived translocation t(4;11) was shown to involve a deletion of the region defined by YAC 13HH4. A leukemia with a deletion on chromosome 11 (q23-q25) was also studied and it was shown that the YAC sequence was unaffected. It was concluded that, with a few exceptions, the translocations at 11q23 in a wide range of acute infant and adult leukemias occur in a common region and may result from a common underlying mechanism.

scholarly journals Active Role of a Human Genomic Insert in Replication of a Yeast Artificial Chromosome

1999 ◽  
Vol 19 (6) ◽  
pp. 4231-4240 ◽  
Author(s):  
Anja J. van Brabant ◽  
Walton L. Fangman ◽  
Bonita J. Brewer
Keyword(s):  
Yeast Artificial Chromosome ◽  
Consensus Sequence ◽  
Artificial Chromosome ◽  
Active Role ◽  
Cell Receptor ◽  
Yeast Cells ◽  
Artificial Chromosomes ◽  
Human T Cell ◽  
T Cell Receptor Beta ◽  
Activation Times

ABSTRACT Yeast artificial chromosomes (YACs) are a common tool for cloning eukaryotic DNA. The manner by which large pieces of foreign DNA are assimilated by yeast cells into a functional chromosome is poorly understood, as is the reason why some of them are stably maintained and some are not. We examined the replication of a stable YAC containing a 240-kb insert of DNA from the human T-cell receptor beta locus. The human insert contains multiple sites that serve as origins of replication. The activity of these origins appears to require the yeast ARS consensus sequence and, as with yeast origins, additional flanking sequences. In addition, the origins in the human insert exhibit a spacing, a range of activation efficiencies, and a variation in times of activation during S phase similar to those found for normal yeast chromosomes. We propose that an appropriate combination of replication origin density, activation times, and initiation efficiencies is necessary for the successful maintenance of YAC inserts.

scholarly journals Motile Properties of Vimentin Intermediate Filament Networks in Living Cells

1998 ◽  
Vol 143 (1) ◽  
pp. 147-157 ◽  
Author(s):  
Miri Yoon ◽  
Robert D. Moir ◽  
Veena Prahlad ◽  
Robert D. Goldman
Keyword(s):  
Intermediate Filament ◽  
Cell Shape ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
A Cell ◽  
Filament Networks ◽  
Green Fluorescent ◽  
Vimentin Intermediate Filament

The motile properties of intermediate filament (IF) networks have been studied in living cells expressing vimentin tagged with green fluorescent protein (GFP-vimentin). In interphase and mitotic cells, GFP-vimentin is incorporated into the endogenous IF network, and accurately reports the behavior of IF. Time-lapse observations of interphase arrays of vimentin fibrils demonstrate that they are constantly changing their configurations in the absence of alterations in cell shape. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Intriguingly, neighboring fibrils within a cell can exhibit different rates and directions of movement, and they often appear to extend or elongate into the peripheral regions of the cytoplasm. In these same regions, short filamentous structures are also seen actively translocating. All of these motile properties require energy, and the majority appear to be mediated by interactions of IF with microtubules and microfilaments.

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