scholarly journals Genome-Wide Analysis Identifies MYND-Domain Protein Mub1 as an Essential Factor for Rpn4 Ubiquitylation

2007 ◽  
Vol 28 (4) ◽  
pp. 1404-1412 ◽  
Author(s):  
Donghong Ju ◽  
Xiaogang Wang ◽  
Haiming Xu ◽  
Youming Xie
Keyword(s):  
Ubiquitin Ligase ◽  
Single Gene ◽  
Genome Wide ◽  
Entire Collection ◽  
In Vitro Reconstitution ◽  
Negative Feedback Circuit ◽  
E2 Enzymes ◽  
Yeast Mutants ◽  
Single Gene Deletion

ABSTRACT The proteasome homeostasis in Saccharomyces cerevisiae is regulated by a negative feedback circuit in which the Rpn4 transcription factor upregulates the proteasome genes and is rapidly degraded by the proteasome. Previous work has identified Ubr2 and Rad6 as the cognate E3 and E2 enzymes for Rpn4 ubiquitylation. However, our recent attempts to ubiquitylate Rpn4 using purified Ubr2 and Rad6 proteins in a reconstitution system have been unsuccessful, suggesting that an additional factor is required for Rpn4 ubiquitylation. Here, we screened the entire collection of the single-gene-deletion yeast mutants generated by the Saccharomyces Genome Deletion Project and identified the mub1Δ mutant defective in ubiquitin-dependent degradation of Rpn4. An in vitro reconstitution ubiquitylation assay confirms that Mub1 is the missing factor for Rpn4 ubiquitylation. We further show that Mub1 directly interacts with Ubr2 and Rpn4. The MYND domain of Mub1 may play an important role in Rpn4 ubiquitylation. Interestingly, Mub1 itself is a short-lived protein and its degradation is dependent on the Ubr2/Rad6 ubiquitin ligase. Together, these data suggest that Mub1 and Ubr2 cooperate to transfer ubiquitin to Rpn4 from Rad6 and that Mub1 may switch from a partner to a substrate of the Ubr2/Rad6 ubiquitin ligase.

scholarly journals Genome-wide karyomapping accurately identifies the inheritance of single-gene defects in human preimplantation embryos in vitro

2014 ◽  
Vol 16 (11) ◽  
pp. 838-845 ◽  
Author(s):  
Senthilkumar A. Natesan ◽  
Alex J. Bladon ◽  
Serdar Coskun ◽  
Wafa Qubbaj ◽  
Renata Prates ◽  
...  
Keyword(s):  
Single Gene ◽  
Preimplantation Embryos ◽  
Genome Wide ◽  
Gene Defects ◽  
Human Preimplantation Embryos

scholarly journals Sulfate import in Salmonella Typhimurium impacts bacterial aggregation and the respiratory burst in human neutrophils

2021 ◽  
Author(s):  
T. L. Westerman ◽  
M. K. Sheats ◽  
J. R. Elfenbein
Keyword(s):  
Respiratory Burst ◽  
Gene Deletion ◽  
Single Gene ◽  
Protein A ◽  
Human Neutrophils ◽  
Deletion Mutants ◽  
Flagellar Motility ◽  
Neutrophil Respiratory Burst ◽  
Single Gene Deletion

During enteric salmonellosis, neutrophil generated reactive oxygen species alter the gut microenvironment favoring survival of Salmonella Typhimurium. While the type-3 secretion system-1 (T3SS-1) and flagellar motility are potent Salmonella Typhimurium agonists of the neutrophil respiratory burst in vitro, neither of these pathways alone are responsible for stimulation of a maximal respiratory burst. In order to identify Salmonella Typhimurium genes that impact the magnitude of the neutrophil respiratory burst, we performed a two-step screen of defined mutant libraries in co-culture with human neutrophils. We first screened Salmonella Typhimurium mutants lacking defined genomic regions and then tested single gene deletion mutants representing particular regions under selection. A subset of single gene deletion mutants were selected for further investigation. Mutants in four genes, STM1696 (sapF), STM2201 (yeiE), STM2112 (wcaD), and STM2441 (cysA), induced an attenuated respiratory burst. We linked the altered respiratory burst to reduced T3SS-1 expression and/or altered flagellar motility for two mutants (ΔSTM1696 and ΔSTM2201). The ΔSTM2441 mutant, defective for sulfate transport, formed aggregates in minimal media and adhered to surfaces in rich media, suggesting a role for sulfur homeostasis in regulation of aggregation/adherence. We linked the aggregation/adherence phenotype of the ΔSTM2441 mutant to biofilm-associated protein A and flagellins and hypothesize that aggregation caused the observed reduction in the magnitude of the neutrophil respiratory burst. Our data demonstrate that Salmonella Typhimurium has numerous mechanisms to limit the magnitude of the neutrophil respiratory burst. These data further inform our understanding of how Salmonella may alter human neutrophil antimicrobial defenses.

scholarly journals Genome-wide analysis of cell-gene interactions

2017 ◽  
Author(s):  
S. Cardinale
Keyword(s):  
Single Gene ◽  
Rna Stability ◽  
Cellular Functions ◽  
Synthetic Genes ◽  
E Coli ◽  
Cell Functions ◽  
Specific Effects ◽  
Genome Wide ◽  
Cell Gene ◽  
Single Gene Deletion

AbstractThe study presents an analysis of how different cellular functions link cell size to the expression of synthetic genes inE. coli. The Size-Expression interaction was mapped with a two-gene genetic probe across 3800 single-gene deletion strains. Through regression analysis, expression-specific effects and gene-specific effects were derived from size effects and generic expression effects, respectively. The entire compendium of cell functions broadly mapped to four systems of distinct primary influence on the Size-Expression map. Specifically, membrane structural components primarily affected size, whereas protein and RNA stability primarily affected gene expression. In addition, major Size-Expression shifts showed no substantial gene-specific effects unless they were mediated by key components of the protein synthesis apparatus.Subject Category:Synthetic Biology

scholarly journals SKP1-like protein, CrSKP1-e, interacts with pollen-specific F-box proteins and assembles into SCF-type E3 complex in ‘Wuzishatangju’ (Citrus reticulata Blanco) pollen

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10578
Author(s):  
Yi Ren ◽  
Qingzhu Hua ◽  
Jiayan Pan ◽  
Zhike Zhang ◽  
Jietang Zhao ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Citrus Reticulata ◽  
S Locus ◽  
Binding Assays ◽  
In Vitro Binding ◽  
Genome Wide ◽  
Ubiquitin Ligase Complex ◽  
Vitro Binding

S-ribonuclease (S-RNase)-based self-incompatibility (SI) mechanisms have been extensively studied in Solanaceae, Rosaceae and Plantaginaceae. S-RNase-based SI is controlled by two closely related genes, S-RNase and S-locus F-box (SLF), located at a polymorphic S-locus. In the SI system, the SCF-type (SKP1-CUL1-F-box-RBX1) complex functions as an E3 ubiquitin ligase complex for ubiquitination of non-self S-RNase. Pummelo (Citrus grandis) and several mandarin cultivars are suggested to utilize an S-RNase-based SI system. However, the molecular mechanism of the non-S-factors involved in the SI reaction is not straightforward in Citrus. To investigate the SCF-type E3 complex responsible for the SI reaction in mandarin, SLF, SKP1-like and CUL1 candidates potentially involved in the SI reaction of ‘Wuzishatangju’ (Citrus reticulata Blanco) were identified based on the genome-wide identification and expression analyses. Sixteen pollen-specific F-box genes (CrFBX1-CrFBX16), one pollen-specific SKP1-like gene (CrSKP1-e) and two CUL1 genes (CrCUL1A and CrCUL1B) were identified and cloned from ‘Wuzishatangju’. Yeast two-hybrid (Y2H) and in vitro binding assays showed that five CrFBX proteins could bind to CrSKP1-e, which is an ortholog of SSK1 (SLF-interacting-SKP1-like), a non-S-factor responsible for the SI reaction. Luciferase complementation imaging (LCI) and in vitro binding assays also showed that CrSKP1-e interacts with the N-terminal region of both CrCUL1A and CrCUL1B. These results indicate that CrSKP1-e may serve as a functional member of the SCF-type E3 ubiquitin ligase complex in ‘Wuzishatangju’.

scholarly journals Competitiveness for Nodule Colonization in Sinorhizobium meliloti: Combined In Vitro -Tagged Strain Competition and Genome-Wide Association Analysis

mSystems ◽  
2021 ◽  
Author(s):  
Agnese Bellabarba ◽  
Giovanni Bacci ◽  
Francesca Decorosi ◽  
Erki Aun ◽  
Elisa Azzarello ◽  
...  
Keyword(s):  
Social Interaction ◽  
Association Analysis ◽  
Sinorhizobium Meliloti ◽  
Single Gene ◽  
A Priori ◽  
Genome Wide Association ◽  
Strain Competition ◽  
Genome Wide ◽  
Competitive Pattern

Decoding the competitive pattern that occurs in the rhizosphere is challenging in the study of bacterial social interaction strategies. To date, the single-gene approach has mainly been used to uncover the bases of nodulation, but there is still a knowledge gap regarding the main features that a priori characterize rhizobial strains able to outcompete indigenous rhizobia.

scholarly journals Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome

2019 ◽  
Author(s):  
Sezen Meydan ◽  
James Marks ◽  
Dorota Klepacki ◽  
Virag Sharma ◽  
Pavel V. Baranov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Translation Initiation ◽  
Single Gene ◽  
Ribosome Profiling ◽  
Genome Wide ◽  
Translation Start ◽  
Alternative Translation

SUMMARYThe use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes but, strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at an internal in-frame and out-of-frame start sites, can be functionally important and contribute to the ‘alternative’ bacterial proteome. The internal start sites my also play regulatory roles in gene expression.

Natural selection defines the cellular complexity

2015 ◽  
Author(s):  
Han Chen ◽  
Xionglei He
Keyword(s):  
Natural Selection ◽  
Molecular System ◽  
Single Gene ◽  
Functional Space ◽  
Simple Expression ◽  
A Cell ◽  
Yeast Mutants ◽  
Protein Modules ◽  
Single Gene Deletion ◽  
Trait Correlation

Current biology is perplexed by the lack of a theoretical framework for understanding the organization principles of the molecular system within a cell. Here we first studied growth rate, one of the seemingly most complex cellular traits, using functional data of yeast single-gene deletion mutants. We observed nearly one thousand expression informative genes (EIGs) whose expression levels are linearly correlated to the trait within an unprecedentedly large functional space. A simple model considering six EIG-formed protein modules revealed a variety of novel mechanistic insights, and also explained ~50% of the variance of cell growth rates measured by Bar-seq technique for over 400 yeast mutants (Pearson's R = 0.69), a performance comparable to the microarray-based (R = 0.77) or colony-size-based (R = 0.66) experimental approach. We then applied the same strategy to 501 morphological traits of the yeast and achieved successes in most fitness-coupled traits each with hundreds of trait-specific EIGs. Surprisingly, there is no any EIG found for most fitness-uncoupled traits, indicating that they are controlled by super-complex epistases that allow no simple expression-trait correlation. Thus, EIGs are recruited exclusively by natural selection, which builds a rather simple functional architecture for fitness-coupled traits, and the endless complexity of a cell lies primarily in its fitness-uncoupled features.

scholarly journals Transcriptional and Post-Transcriptional Polar Effects in Bacterial Gene Deletion Libraries

mSystems ◽  
2021 ◽  
Author(s):  
André Mateus ◽  
Malay Shah ◽  
Johannes Hevler ◽  
Nils Kurzawa ◽  
Jacob Bobonis ◽  
...  
Keyword(s):  
Gene Function ◽  
Gene Deletion ◽  
Single Gene ◽  
Bacterial Gene ◽  
Genome Wide ◽  
Single Gene Deletion ◽  
Deletion Libraries

Single-gene deletion libraries have allowed genome-wide characterization of gene function and interactions. While each mutant intends to disrupt the function of a single gene, it can unintentionally target other genes, such as those located in the same operon as the deletion.

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