scholarly journals Structural and Functional Characterization of the N Terminus of Schizosaccharomyces pombe Cwf10

2013 ◽  
Vol 12 (11) ◽  
pp. 1472-1489 ◽  
Author(s):  
S. Brent Livesay ◽  
Scott E. Collier ◽  
Danny A. Bitton ◽  
Jürg Bähler ◽  
Melanie D. Ohi
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Functional Characterization ◽  
Elongation Factor ◽  
Mrna Splicing ◽  
Multiple Roles ◽  
Translation Elongation ◽  
Content Type ◽  
Eukaryotic Translation ◽  
Spliceosome Activation

ABSTRACT The spliceosome is a dynamic macromolecular machine that catalyzes the removal of introns from pre-mRNA, yielding mature message. Schizosaccharomyces pombe Cwf10 (homolog of Saccharomyces cerevisiae Snu114 and human U5-116K), an integral member of the U5 snRNP, is a GTPase that has multiple roles within the splicing cycle. Cwf10/Snu114 family members are highly homologous to eukaryotic translation elongation factor EF2, and they contain a conserved N-terminal extension (NTE) to the EF2-like portion, predicted to be an intrinsically unfolded domain. Using S. pombe as a model system, we show that the NTE is not essential, but cells lacking this domain are defective in pre-mRNA splicing. Genetic interactions between cwf10 -Δ NTE and other pre-mRNA splicing mutants are consistent with a role for the NTE in spliceosome activation and second-step catalysis. Characterization of Cwf10-NTE by various biophysical techniques shows that in solution the NTE contains regions of both structure and disorder. The first 23 highly conserved amino acids of the NTE are essential for its role in splicing but when overexpressed are not sufficient to restore pre-mRNA splicing to wild-type levels in cwf10 -Δ NTE cells. When the entire NTE is overexpressed in the cwf10 -Δ NTE background, it can complement the truncated Cwf10 protein in trans , and it immunoprecipitates a complex similar in composition to the late-stage U5.U2/U6 spliceosome. These data show that the structurally flexible NTE is capable of independently incorporating into the spliceosome and improving splicing function, possibly indicating a role for the NTE in stabilizing conformational rearrangements during a splice cycle.

scholarly journals Isolation and characterization of monoclonal antibodies against eukaryotic translation elongation factor 1A

2003 ◽  
Vol 19 (3) ◽  
pp. 231-237
Author(s):  
A. P. Pogrebnaya ◽  
N. V. Markeyeva ◽  
V. V. Lisogubov ◽  
A. M. Fridberg ◽  
S. I. Zabashnyi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Elongation Factor ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Eukaryotic Translation ◽  
Isolation And Characterization ◽  
Translation Elongation Factor 1A

scholarly journals Morphological and Molecular Characterization of Exophiala polymorpha sp. nov. Isolated from Sporotrichoid Lymphocutaneous Lesions in a Patient with Myasthenia Gravis

2015 ◽  
Vol 53 (9) ◽  
pp. 2816-2822 ◽  
Author(s):  
Lee K. Yong ◽  
Nathan P. Wiederhold ◽  
Deanna A. Sutton ◽  
Marcelo Sandoval-Denis ◽  
Jonathan R. Lindner ◽  
...  
Keyword(s):  
Myasthenia Gravis ◽  
Molecular Characterization ◽  
Elongation Factor ◽  
Internal Transcribed Spacer Region ◽  
Translation Elongation ◽  
Content Type ◽  
Elongation Factor 1 Alpha ◽  
Morphological And Molecular Characterization

Exophialaspecies are capable of causing cutaneous and subcutaneous infections in immunocompromised patients. AnExophialaisolate was cultured from a biopsy specimen of a lesion on the forearm of a patient with myasthenia gravis. The patient also had lesions on the palm and distal aspects of the hand, which were successfully treated with a long-term course of itraconazole. A detailed morphological and molecular characterization of the isolate was undertaken. Phylogenetic analysis of the internal transcribed spacer region and portions of the β-tubulin and translation elongation factor 1-alpha genes indicated that the isolate was a novel species closely related to but genetically distinct from species within theExophiala spiniferaclade; the nameExophiala polymorphasp. nov. is proposed. Morphologically,E. polymorphamost closely resemblesE. xenobioticabut it differs in possessing phialides bearing prominent, wide collarettes, and it does not produce chlamydospores.

scholarly journals Characterization of Glycoside Hydrolase Family 5 Proteins in Schizosaccharomyces pombe

2010 ◽  
Vol 9 (11) ◽  
pp. 1650-1660 ◽  
Author(s):  
Encarnación Dueñas-Santero ◽  
Ana Belén Martín-Cuadrado ◽  
Thierry Fontaine ◽  
Jean-Paul Latgé ◽  
Francisco del Rey ◽  
...  
Keyword(s):  
Cell Wall ◽  
Schizosaccharomyces Pombe ◽  
Protein Characterization ◽  
Wall Material ◽  
Glycoside Hydrolase Family ◽  
Content Type ◽  
Hydrolysis Of ◽  
Controlled Hydrolysis ◽  
Hydrolase Family

ABSTRACT In yeast, enzymes with β-glucanase activity are thought to be necessary in morphogenetic events that require controlled hydrolysis of the cell wall. Comparison of the sequence of the Saccharomyces cerevisiae exo-β(1,3)-glucanase Exg1 with the Schizosaccharomyces pombe genome allowed the identification of three genes that were named exg1 + (locus SPBC1105.05), exg2 + (SPAC12B10.11), and exg3 + (SPBC2D10.05). The three proteins have different localizations: Exg1 is secreted to the periplasmic space, Exg2 is a membrane protein, and Exg3 is a cytoplasmic protein. Characterization of the biochemical activity of the proteins indicated that Exg1 and Exg3 are active only against β(1,6)-glucans while no activity was detected for Exg2. Interestingly, Exg1 cleaves the glucans with an endohydrolytic mode of action. exg1 + showed periodic expression during the cell cycle, with a maximum coinciding with the septation process, and its expression was dependent on the transcription factor Sep1. The Exg1 protein localizes to the septum region in a pattern that was different from that of the endo-β(1,3)-glucanase Eng1. Overexpression of Exg2 resulted in an increase in cell wall material at the poles and in the septum, but the putative catalytic activity of the protein was not required for this effect.

Functional analysis of a stable transcription arrest site in the first intron of the murine adenosine deaminase gene

1993 ◽  
Vol 13 (5) ◽  
pp. 2718-2729
Author(s):  
S F Kash ◽  
J W Innis ◽  
A U Jackson ◽  
R E Kellems
Keyword(s):  
Rna Polymerase Ii ◽  
Adenosine Deaminase ◽  
Functional Characterization ◽  
Gel Filtration ◽  
Point Mutations ◽  
Elongation Factor ◽  
Dependent Manner ◽  
First Intron ◽  
Intron 1

Transcription arrest plays a role in regulating the expression of a number of genes, including the murine adenosine deaminase (ADA) gene. We have previously identified two prominent arrest sites at the 5' end of the ADA gene: one in the first exon and one in the first intron (J. W. Innis and R. E. Kellems, Mol. Cell. Biol. 11:5398-5409, 1991). Here we report the functional characterization of the intron 1 arrest site, located 137 to 145 nucleotides downstream of the cap site. We have determined, using gel filtration, that the intron 1 arrest site is a stable RNA polymerase II pause site and that the transcription elongation factor SII promotes read-through at this site. Additionally, the sequence determinants for the pause are located within a 37-bp fragment encompassing this site (+123 to +158) and can direct transcription arrest in an orientation-dependent manner in the context of the ADA and adenovirus major late promoters. Specific point mutations in this region increase or decrease the relative pausing efficiency. We also show that the sequence determinants for transcription arrest can function when placed an additional 104 bp downstream of their natural position.

scholarly journals N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Stanislav Huszár ◽  
Vinayak Singh ◽  
Alica Polčicová ◽  
Peter Baráth ◽  
María Belén Barrio ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Functional Characterization ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Libraries ◽  
Whole Cells ◽  
Encoding Gene

ABSTRACT The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

scholarly journals Survey, Identification, and Characterization of Cylindrocarpon-Like Asexual Morphs in Spanish Forest Nurseries

Plant Disease ◽  
2018 ◽  
Vol 102 (11) ◽  
pp. 2083-2100 ◽  
Author(s):  
Beatriz Mora-Sala ◽  
Ana Cabral ◽  
Maela León ◽  
Carlos Agustí-Brisach ◽  
Josep Armengol ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Elongation Factor ◽  
Its Region ◽  
Phylogenetic Position ◽  
Translation Elongation ◽  
Forest Nurseries ◽  
Asexual Morphs ◽  
Identification And Characterization ◽  
Elongation Factor 1

Cylindrocarpon-like asexual morphs infect herbaceous and woody plants, mainly in agricultural scenarios, but also in forestry systems. The aim of the present study was to characterize a collection of Cylindrocarpon-like isolates recovered from the roots of a broad range of forest hosts from nurseries showing decline by morphological and molecular studies. Between 2009 and 2012, 17 forest nurseries in Spain were surveyed and a total of 103 Cylindrocarpon-like isolates were obtained. Isolates were identified based on DNA sequences of the partial gene regions histone H3 (his3). For the new species, the internal transcribed spacer and intervening 5.8S nrRNA gene (ITS) region, β-tubulin (tub2), and translation elongation factor 1-α (tef1) were also used to determine their phylogenetic position. Twelve species belonging to the genera Cylindrodendrum, Dactylonectria, and Ilyonectria were identified from damaged roots of 15 different host genera. The species C. alicantinum, D. macrodidyma, D. novozelandica, D. pauciseptata, D. pinicola, D. torresensis, I. capensis, I. cyclaminicola, I. liriodendri, I. pseudodestructans, I. robusta, and I. rufa were identified. In addition, two Dactylonectria species (D. hispanica sp. nov. and D. valentina sp. nov.), one Ilyonectria species (I. ilicicola sp. nov.), and one Neonectria species (N. quercicola sp. nov.) are newly described. The present study demonstrates the prevalence of this fungal group associated with seedlings of diverse hosts showing decline symptoms in forest nurseries in Spain.

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