scholarly journals Rapid Production of Gene Replacement Constructs and Generation of a Green Fluorescent Protein-Tagged Centromeric Marker in Aspergillus nidulans

2004 ◽  
Vol 3 (5) ◽  
pp. 1359-1362 ◽  
Author(s):  
Lin Yang ◽  
Leena Ukil ◽  
Aysha Osmani ◽  
Francis Nahm ◽  
Jonathan Davies ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Aspergillus Nidulans ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Gene Replacement ◽  
Deletion Analysis ◽  
Rapid Production ◽  
Promoter Swapping ◽  
Green Fluorescent

ABSTRACT A method to rapidly generate gene replacement constructs by fusion PCR is described for Aspergillus nidulans. The utility of the approach is demonstrated by green fluorescent protein (GFP) tagging of A. nidulans ndc80 to visualize centromeres through the cell cycle. The methodology makes possible large-scale GFP tagging, promoter swapping, and deletion analysis of A. nidulans.

The Fe-Core/Carbon-Shell Ultrafine Nanopowders as Platform for Biomolecules Grafting

2014 ◽  
Vol 1040 ◽  
pp. 194-198
Author(s):  
N.S. Surgutskaya ◽  
P.S. Postnikov ◽  
Alexandra G. Pershina ◽  
A.I. Galanov ◽  
Marina E. Trusova ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Carbon Shell ◽  
Shell Nanoparticles ◽  
Powder Surface ◽  
Metal Precursors ◽  
Covalent Grafting ◽  
Large Scale Synthesis ◽  
Green Fluorescent

The Fe-core/carbon-shell nanopowders are excellent platform for covalent grafting of biomolecules. The large-scale synthesis of Fe-core/carbon-shell nanoparticles via electropulse erosion of metal precursors in hydrocarbons was developed. The green fluorescent protein was covalently attached to the powder surface via diazonium functionalization and further carbodiimide activation.

scholarly journals Conditional Transgenic System for Mouse Aurora A Kinase: Degradation by the Ubiquitin Proteasome Pathway Controls the Level of the Transgenic Protein

2005 ◽  
Vol 25 (12) ◽  
pp. 5270-5281 ◽  
Author(s):  
Tomokazu Fukuda ◽  
Yuji Mishina ◽  
Michael P. Walker ◽  
Richard P. DiAugustine
Keyword(s):  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Aurora A ◽  
Mitotic Kinase ◽  
Mitotic Stage ◽  
Conditional Expression ◽  
Ubiquitin Proteasome ◽  
Green Fluorescent

ABSTRACT Aurora A is a mitotic kinase that localizes to centrosomes. Expression of this protein is normally limited to the mitotic stage (G2-M) of the cell cycle, whereas human cancer cells frequently exhibit overexpression of Aurora A protein regardless of the cell cycle stage. In the present study, Aurora A transgenic mouse lines were generated with a new conditional expression system (cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter-Z-enhanced green fluorescent protein) in order to analyze the function of this protein. Although transcripts for Aurora A were elevated in multiple organs of the transgenic mice, the corresponding protein was not detected in extracts analyzed by immunoblotting. The treatment of transgenic-derived embryonic fibroblasts (MEF) with proteasome inhibitors markedly increased the protein level of transgenic Aurora A, indicating that the transgenic Aurora A protein is readily degraded in normal mouse tissues. Under the exponential growth conditions of MEF cells, transgenic Aurora A was detected within the mitotic stage of the cell cycle and localized to centrosomes. In contrast, the marker of the transgenic promoter (enhanced green fluorescent protein) was continuously expressed throughout the cell cycle, indicating the constitutive transcription of transgenic mRNA. These results indicate that transgenic Aurora A is protected from degradation within G2-M but is immediately degraded after translation in the G1-S stage of the cell cycle. The findings obtained with this transgenic model and derived cells support that the transition from protection to degradation by the ubiquitin proteasome system at the end of mitosis is an important step in controlling the level of Aurora A protein during the cell cycle.

Correlating cell cycle with apoptosis in a cell line expressing a tandem green fluorescent protein substrate specific for group II caspases

Cytometry ◽  
2001 ◽  
Vol 45 (3) ◽  
pp. 225-234 ◽  
Author(s):  
Christopher J. Donahue ◽  
Maxine Santoro ◽  
Donald Hupe ◽  
Jay M. Jones ◽  
Brian Pollok ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Cell Line ◽  
Fluorescent Protein ◽  
Protein Substrate ◽  
A Cell ◽  
Group Ii ◽  
Green Fluorescent

scholarly journals The Sudden Recruitment of γ-Tubulin to the Centrosome at the Onset of Mitosis and Its Dynamic Exchange Throughout the Cell Cycle, Do Not Require Microtubules

1999 ◽  
Vol 146 (3) ◽  
pp. 585-596 ◽  
Author(s):  
Alexey Khodjakov ◽  
Conly L. Rieder
Keyword(s):  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Fusion ◽  
Protein Fusion ◽  
Green Fluorescent ◽  
Dynamic Exchange ◽  
Two Populations

γ-Tubulin is a centrosomal component involved in microtubule nucleation. To determine how this molecule behaves during the cell cycle, we have established several vertebrate somatic cell lines that constitutively express a γ-tubulin/green fluorescent protein fusion protein. Near simultaneous fluorescence and DIC light microscopy reveals that the amount of γ-tubulin associated with the centrosome remains relatively constant throughout interphase, suddenly increases during prophase, and then decreases to interphase levels as the cell exits mitosis. This mitosis-specific recruitment of γ-tubulin does not require microtubules. Fluorescence recovery after photobleaching (FRAP) studies reveal that the centrosome possesses two populations of γ-tubulin: one that turns over rapidly and another that is more tightly bound. The dynamic exchange of centrosome-associated γ-tubulin occurs throughout the cell cycle, including mitosis, and it does not require microtubules. These data are the first to characterize the dynamics of centrosome-associated γ-tubulin in vertebrate cells in vivo and to demonstrate the microtubule-independent nature of these dynamics. They reveal that the additional γ-tubulin required for spindle formation does not accumulate progressively at the centrosome during interphase. Rather, at the onset of mitosis, the centrosome suddenly gains the ability to bind greater than three times the amount of γ-tubulin than during interphase.

scholarly journals Roles of NUDE and NUDF Proteins of Aspergillus nidulans: Insights from Intracellular Localization and Overexpression Effects

2003 ◽  
Vol 14 (3) ◽  
pp. 871-888 ◽  
Author(s):  
Vladimir P. Efimov
Keyword(s):  
Green Fluorescent Protein ◽  
Aspergillus Nidulans ◽  
Heavy Chain ◽  
Fluorescent Protein ◽  
Specific Interaction ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Dynein Heavy Chain ◽  
Terminal Domain ◽  
Green Fluorescent

The NUDF protein of the filamentous fungus Aspergillus nidulans functions in the cytoplasmic dynein pathway. It binds several proteins, including the NUDE protein. Green fluorescent protein-tagged NUDF and NUDA (dynein heavy chain) localize to linearly moving dashes (“comets”) that coincide with microtubule ends. Herein, deletion of the nudE gene did not eliminate the comets of NUDF and NUDA, but affected the behavior of NUDA. Comets were also observed with the green fluorescent protein-tagged NUDE and its nonfunctional C-terminal domain. In addition, overexpressed NUDA and NUDE accumulated in specks that were either immobile or bounced randomly. Neither comets nor specks were observed with the functional N-terminal domain of NUDE, indicating that these structures are not essential for NUDE function. Furthermore, NUDF overproduction totally suppressed deletion of the nudEgene. This implies that the function of NUDE is secondary to that of NUDF. Unexpectedly, NUDF overproduction inhibited one conditionalnudA mutant and all tested apsA mutants. An allele-specific interaction between the nudF andnudA genes is consistent with a direct interaction between NUDF and dynein heavy chain. Because APSA and its yeast homolog Num1p are cortical proteins, an interaction between thenudF and apsA genes suggests a role for NUDF at the cell cortex.

scholarly journals Green Fluorescent Protein-Dal80p Illuminates up to 16 Distinct Foci That Colocalize with and Exhibit the Same Behavior as Chromosomal DNA Proceeding through the Cell Cycle of Saccharomyces cerevisiae

2001 ◽  
Vol 183 (15) ◽  
pp. 4636-4642 ◽  
Author(s):  
MacKenzie Distler ◽  
Ajit Kulkarni ◽  
Rajendra Rai ◽  
Terrance G. Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Green Fluorescent Protein ◽  
Leucine Zipper ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Live Cells ◽  
Nitrogen Catabolite Repression ◽  
Chromosomal Dna ◽  
Green Fluorescent

ABSTRACT Four GATA family DNA binding proteins mediate nitrogen catabolite repression-sensitive transcription in Saccharomyces cerevisiae. Gln3p and Gat1p are transcriptional activators, while Dal80p and Deh1p repress Gln3p- and Gat1p-mediated transcription by competing with these activators for binding to DNA. Strong Dal80p binding to DNA is thought to result from C-terminal leucine zipper-mediated dimerization. Many Dal80p binding site-homologous sequences are relatively evenly distributed across the S. cerevisiae genome, raising the possibility that Dal80p might be able to “stain” DNA. We demonstrate that cells containing enhanced green fluorescent protein-Dal80p (EGFP-Dal80p) exhibit up to 16 fluorescent foci that colocalize with DAPI (4′,6′-diamidino-2-phenylindole)-positive material and follow DNA movement through the cell cycle, suggesting that EGFP-Dal80p may indeed be useful for monitoring yeast chromosomes in live cells and in real time.

Sign in / Sign up

Export Citation Format

Share Document