New Insights on the Mechanism of Cyclization in Chromophore Maturation of Wild-Type Green Fluorescence Protein: A Computational Study

2016 ◽  
Vol 120 (24) ◽  
pp. 5386-5394 ◽  
Author(s):  
Yingying Ma ◽  
Hao Zhang ◽  
Qiao Sun ◽  
Sean C. Smith
Keyword(s):  
Computational Study ◽  
Protein A ◽  
Green Fluorescence Protein ◽  
Wild Type ◽  
Green Fluorescence ◽  
Fluorescence Protein

scholarly journals 082 Infection and Transformation of Rhododendron by Agrobacterium tumefaciens Strain B6

HortScience ◽  
1999 ◽  
Vol 34 (3) ◽  
pp. 455C-455 ◽  
Author(s):  
R.R. Tripepi ◽  
M.W. George ◽  
T. Sripo ◽  
S.A. Johnsen ◽  
A.B. Caplan
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetically Modified ◽  
Green Fluorescence Protein ◽  
Plant Genome ◽  
Wild Type ◽  
Bacterial Strains ◽  
Green Fluorescence ◽  
Physical Presence ◽  
Fluorescence Protein ◽  
Polymerase Chain

The objective of this study was to determine if selected strains of Agrobacterium could infect microshoots of Rhododendron catawbiense. Fifteen microshoot stems of R. catawbiense var. album `America', `Joe Paterno', and `Cunningham's White' were inoculated with two drops (about 25 μL) of wild type Agrobacterium tumefaciens strains C58 or B6 or with wild type A. rhizogenes strain E8/73. Five control shoots were inoculated with 1.2 mM KH2PO4 buffer. Microshoots were grown on woody plant medium (WPM) supplemented with 4.9 μM 2iP. Six weeks after inoculation galls that formed were excised from the microshoots and placed on WPM that lacked plant growth regulators but contained 300 mg·L-1 cefotaxime. In another study, these wild-type bacterial strains were genetically modified by inserting the pBINm-gfp5-ER plasmid, which contained genes coding for NPTII and green fluorescence protein (GFP), into the bacteria. These modified strains were inoculated on 15 stems of the three rhododendron cultivars and one variety. Calluses that formed were excised, placed on basal WPM with cefotaxime, and allowed to proliferate. Wild type C58 induced galls to form on `Joe Paterno', R.c. album, and `Cunningham's White' stems, whereas wild type B6 caused galls to form only on the latter two types of rhododendron. Wild-type E8/73 failed to induce gall formation on the rhododendrons. Only genetically modified B6 caused galls to form on only `Cunningham's White' microshoots (seven of 15 inoculated stems). Three of these galls fluoresced green under ultraviolet light. Physical presence of the NPTII and GFP genes in the plant genome was determined by polymerase chain reaction. This study demonstrated that R. catawbiense is susceptible to Agrobacterium infection, and this plant can be genetically transformed.

scholarly journals Kinesin-1 and dynein at the nuclear envelope mediate the bidirectional migrations of nuclei

2010 ◽  
Vol 191 (1) ◽  
pp. 115-128 ◽  
Author(s):  
Heidi N. Fridolfsson ◽  
Daniel A. Starr
Keyword(s):  
Nuclear Envelope ◽  
Green Fluorescence Protein ◽  
Time Lapse ◽  
Nuclear Migration ◽  
Differential Interference Contrast ◽  
Wild Type ◽  
Green Fluorescence ◽  
Fluorescence Protein ◽  
Bidirectional Movement ◽  
Kinetics Of

Kinesin-1 and dynein are recruited to the nuclear envelope by the Caenorhabditis elegans klarsicht/ANC-1/Syne homology (KASH) protein UNC-83 to move nuclei. The mechanisms of how these motors are coordinated to mediate nuclear migration are unknown. Time-lapse differential interference contrast and fluorescence imaging of embryonic hypodermal nuclear migration events were used to characterize the kinetics of nuclear migration and determine microtubule dynamics and polarity. Wild-type nuclei display bidirectional movements during migration and are also able to roll past cytoplasmic granules. unc-83, unc-84, and kinesin-1 mutants have severe nuclear migration defects. Without dynein, nuclear migration initiates normally but lacks bidirectional movement and shows defects in nuclear rolling, implicating dynein in resolution of cytoplasmic roadblocks. Microtubules are highly dynamic during nuclear migration. EB1::green fluorescence protein imaging demonstrates that microtubules are polarized in the direction of nuclear migration. This organization of microtubules fits with our model that kinesin-1 moves nuclei forward and dynein functions to move nuclei backward for short stretches to bypass cellular roadblocks.

scholarly journals Forespore Expression and Processing of the SigE Transcription Factor in Wild-Type and Mutant Bacillus subtilis

1998 ◽  
Vol 180 (7) ◽  
pp. 1673-1681 ◽  
Author(s):  
Jingliang Ju ◽  
Tingqiu Luo ◽  
W. G. Haldenwang
Keyword(s):  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Green Fluorescence Protein ◽  
Distal Portion ◽  
Protein Product ◽  
Wild Type ◽  
Green Fluorescence ◽  
Selective Degradation ◽  
Fluorescence Protein ◽  
A Site

ABSTRACT ςE is a mother cell-specific transcription factor of sporulating Bacillus subtilis that is derived from an inactive precursor protein (pro-ςE). To examine the process that prevents ςE activity from developing in the forespore, we fused the ςE structural gene (sigE) to forespore-specific promoters (PdacF and PspoIIIG ), placed these fusions at sites on the B. subtilis chromosome which translocate into the forespore either early or late, and used Western blot analysis to monitor SigE accumulation and pro-ςE processing. sigE alleles, placed at sites which entered the forespore early, were found to generate more protein product than the same fusion placed at a late entering site. SigE accumulation and processing in the forespore were enhanced by null mutations in spoIIIE, a gene whose product is essential for translocation of the distal portion of the B. subtilischromosome into the forespore. In other experiments, a chimera of pro-ςE and green fluorescence protein, previously shown to be unprocessed if it is synthesized within the forespore, was found to be processed in this compartment if coexpressed with the gene for the pro-ςE-processing enzyme, SpoIIGA. The need forspoIIGA coexpression is obviated in the absence of SpoIIIE. We interpret these results as evidence that selective degradation of both SigE and SpoIIGA prevent mature ςE from accumulating in the forespore compartment of wild-type B. subtilis. Presumably, a gene(s) located at a site that is distal to the origin of chromosome transfer is responsible for this phenomenon when it is translocated and expressed in the forespore.

scholarly journals Expansion of EasyClone-MarkerFree toolkit for Saccharomyces cerevisiae genome with new integration sites

2021 ◽  
Author(s):  
Mahsa Babaei ◽  
Luisa Sartori ◽  
Alexey Karpukhin ◽  
Dmitrii Abashkin ◽  
Elena Matrosova ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Recombinant Dna ◽  
Green Fluorescence Protein ◽  
Cellular Growth ◽  
Green Fluorescence ◽  
Biotechnological Production ◽  
Yeast Saccharomyces Cerevisiae ◽  
Integration Sites ◽  
Fluorescence Protein ◽  
Integration Efficiency

Abstract Biotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.

Establishment of an Embryotoxicity Assay with Green Fluorescence Protein-expressing Embryonic Cell-derived Cardiomyocytes

1999 ◽  
Vol 27 (3) ◽  
pp. 471-484 ◽  
Author(s):  
Susanne Bremer ◽  
Maaike Van Dooren ◽  
Martin Paparella ◽  
Eugen Kossolov ◽  
Bernd Fleischmann ◽  
...  
Keyword(s):  
Green Fluorescence Protein ◽  
Embryonic Cell ◽  
Green Fluorescence ◽  
Fluorescence Protein

scholarly journals Fertility of mice receiving vitrified adult mouse ovaries

Reproduction ◽  
2006 ◽  
Vol 131 (4) ◽  
pp. 681-687 ◽  
Author(s):  
Toshio Hani ◽  
Takanori Tachibe ◽  
Saburo Shingai ◽  
Nobuo Kamada ◽  
Otoya Ueda ◽  
...  
Keyword(s):  
Germ Cells ◽  
Adult Mouse ◽  
Green Fluorescence Protein ◽  
Green Fluorescence ◽  
Experimental Animals ◽  
Immature Mouse ◽  
Estrous Cyclicity ◽  
Fluorescence Protein ◽  
High Degree ◽  
Old Mice

Cryopreservation of the ovaries is a useful technology for preservation of germ cells from experimental animals, because if the female founder is infertile or has mutated mitochondrial DNA, preservation of female germ cells is necessary. Although it is possible to cryopreserve immature mouse ovaries with a high degree of viability by vitrification with a mixture of several cryoprotectants, the viability of cryopreserved adult mouse ovaries is still unknown. Here, we investigated the viability of mouse ovaries at various ages after cryopreservation by vitrification techniques. Donor ovaries were collected from 10-day-, 4-week-, 10-week- and 7-month-old, female, nulliparous, green fluorescence protein (GFP)-transgenic mice and cryopreserved by vitrification. The vitrified-warmed ovaries were orthotopically transplanted to 4- or 10-week-old mice. GFP-positive pups were obtained in all experimental groups. In the 4-week-old recipients, the percentages of GFP-positive pups among the total pups from recipients transplanted with ovaries of 10-day-, 4-week-, 10-week- and 7-month-old donors were 44%, 9%, 12% and 4% respectively. In the 10-week-old recipients, the percentages of GFP-positive pups among the total pups from recipients transplanted with ovaries of 10-day-, 4-week-, 10-week- and 7-month-old donors were 36%, 16%, 2% and 9% respectively. Furthermore, GFP-positive pups also were obtained from recipients transplanted with ovaries of donors without normal estrous cyclicity. Our results indicate that cryopreservation of mouse ovaries by vitrification is a useful method for the preservation of female germ cells from mice of various ages.

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