scholarly journals An improved auxin-inducible degron system for fission yeast

2021 ◽  
Author(s):  
Xiao-Ran Zhang ◽  
Lei Zhao ◽  
Fang Suo ◽  
Yadong Gao ◽  
Qingcui Wu ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Binding Pocket ◽  
Essential Genes ◽  
Biological Research ◽  
Auxin Signaling ◽  
Loss Of Function ◽  
Enhance Efficiency ◽  
F Box Protein ◽  
Signaling Components ◽  
Control Protein

Abstract Conditional degron technologies, which allow a protein of interest to be degraded in an inducible manner, are important tools for biological research, and are especially useful for creating conditional loss-of-function mutants of essential genes. The auxin-inducible degron (AID) technology, which utilizes plant auxin signaling components to control protein degradation in nonplant species, is a widely used small-molecular-controlled degradation method in yeasts and animals. However, the currently available AID systems still have room for further optimization. Here, we have improved the AID system for the fission yeast Schizosaccharomyces pombe by optimizing all three components: the AID degron, the small-molecule inducer, and the inducer-responsive F-box protein. We chose a 36-amino-acid sequence of the Arabidopsis IAA17 protein as the degron and employed three tandem copies of it to enhance efficiency. To minimize undesirable side effects of the inducer, we adopted a bulky analog of auxin, 5-adamantyl-IAA, and paired it with the F-box protein OsTIR1 that harbors a mutation (F74A) at the auxin-binding pocket. 5-adamantyl-IAA, when utilized with OsTIR1-F74A, is effective at concentrations thousands of times lower than auxin used in combination with wild-type OsTIR1. We tested our improved AID system on 10 essential genes and achieved inducible lethality for all of them, including ones that could not be effectively inactivated using a previously published AID system. Our improved AID system should facilitate the construction of conditional loss-of-function mutants in fission yeast.

scholarly journals An improved auxin-inducible degron system for fission yeast

2021 ◽  
Author(s):  
Xiao-Ran Zhang ◽  
Lei Zhao ◽  
Fang Suo ◽  
Yadong Gao ◽  
Qingcui Wu ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Binding Pocket ◽  
Essential Genes ◽  
Biological Research ◽  
Auxin Signaling ◽  
Loss Of Function ◽  
Enhance Efficiency ◽  
F Box Protein ◽  
Signaling Components ◽  
Control Protein

ABSTRACTConditional degron technologies, which allow a protein of interest to be degraded in an inducible manner, are important tools for biological research, and are especially useful for creating conditional loss-of-function mutants of essential genes. The auxin-inducible degron (AID) technology, which utilizes plant auxin signaling components to control protein degradation in non-plant species, is a widely used small-molecular-controlled degradation method in yeasts and animals. However, the currently available AID systems still have room for further optimization. Here, we have improved the AID system for the fission yeast Schizosaccharomyces pombe by optimizing all three components: the AID degron, the small-molecule inducer, and the inducer-responsive F-box protein. We chose a 36-amino-acid sequence of the Arabidopsis IAA17 protein as the degron and employed three tandem copies of it to enhance efficiency. To minimize undesirable side effects of the inducer, we adopted a bulky analog of auxin, 5-adamantyl-IAA, and paired it with the F-box protein OsTIR1 that harbors a mutation (F74A) at the auxin-binding pocket. 5-adamantyl-IAA, when utilized with OsTIR1-F74A, is effective at concentrations thousands of times lower than auxin used in combination with wild-type OsTIR1. We tested our improved AID system on 10 essential genes and achieved inducible lethality for all of them, including ones that could not be effectively inactivated using a previously published AID system. Our improved AID system should facilitate the construction of conditional loss-of-function mutants in fission yeast.

scholarly journals The Hoja loca1 Mutant and AUX/IAA Function in Maize

2020 ◽  
Author(s):  
A. E. Richardson ◽  
A. Sluis ◽  
S. Hake
Keyword(s):  
Functional Redundancy ◽  
Vital Role ◽  
Land Plants ◽  
Auxin Signaling ◽  
Organ Development ◽  
Loss Of Function ◽  
Plant Organ ◽  
Species Specific ◽  
Signaling Components

AbstractAuxin plays a vital role in plant organ development, influencing organ initiation and patterning across all axes. The diversity in auxin patterning results from changes in the activities and expression of auxin signaling components, including the AUX/IAA repressors. Higher land plants have multigene AUX/IAA families, which leads to functional redundancy and a lack of phenotype in loss of function mutants. Instead, dominant mutations, which prevent AUX/IAA degradation in response to auxin, have highlighted the importance of these proteins in organ development. Here we report a new dominant AUX/IAA mutant in maize, Hoja loca1 (Oja). Oja has a mutation in the degron motif of ZmIAA28 and affects aerial organ initiation and medio-lateral patterning in the leaf. These phenotypes contrast with other maize AUX/IAA mutants that affect the root or inflorescence only. Oja illustrates the role of auxin signaling in the tight coordination of phytomer unit development and provides evidence of species-specific sub-functionalization of the AUX/IAAs.One Line SummaryThe maize AUX/IAA ZmIAA38 is involved in phytomer coordination, aerial organ initiation and mediolateral patterning, and illustrates species specific sub-functionalization of the AUX/IAAs.

scholarly journals The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Minkyung Choi ◽  
Jae-Young Yun ◽  
Jun-Hyuk Kim ◽  
Jin-Soo Kim ◽  
Sang-Tae Kim
Keyword(s):  
Arabidopsis Thaliana ◽  
Cytidine Deaminase ◽  
Biological Research ◽  
Loss Of Function ◽  
Nucleotide Substitutions ◽  
Viral Origin ◽  
Nonsense Mutations ◽  
Base Editing ◽  
Low Efficiency ◽  
Viable System

AbstractCRISPR/Cas9-mediated genome editing is an important and versatile technology in modern biological research. Recent advancements include base-editing CRISPR tools that enable targeted nucleotide substitutions using a fusion protein comprising a nickase variant of Cas9 and a base deaminase. Improvements in base editing efficiencies and inheritable of edited loci need to be made to make CRISPR a viable system in plants. Here, we report efficiency of cytosine base editors (CBEs) in Arabidopsis thaliana by applying the strong endogenous RPS5a promoter to drive the expression of nickase Cas9 and either rAPOBEC1 from rat (BE3) or the PmCDA1 activation-induced cytidine deaminase from sea lamprey (AIDv2). Compared with the strong heterologous CaMV35S promoter of viral origin, the RPS5a promoter improved CBE efficiency by 32% points with the number of T1 plants showing over 50% conversion ratio when the LFY gene was targeted. CBE induced nonsense mutations in LFY via C-to-T conversion, which resulted in loss-of-function lfy phenotypes; defects in LFY function were associated with the targeted base substitutions. Our data suggest that optimal promoter choice for CBE expression may affect base-editing efficiencies in plants. The results provide a strategy to optimize low-efficiency base editors and demonstrate their applicability for functional assays and trait development in crop research.

scholarly journals An integrated approach to unravel a crucial structural property required for the function of the insect steroidogenic Halloween protein Noppera-bo

2020 ◽  
Vol 295 (20) ◽  
pp. 7154-7167 ◽  
Author(s):  
Kotaro Koiwai ◽  
Kazue Inaba ◽  
Kana Morohashi ◽  
Sora Enya ◽  
Reina Arai ◽  
...  
Keyword(s):  
Structural Characteristics ◽  
Integrated Approach ◽  
Binding Pocket ◽  
Loss Of Function ◽  
Tertiary Structures ◽  
Ecdysteroid Biosynthesis ◽  
17Β Estradiol ◽  
Unique Amino Acid

Ecdysteroids are the principal steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis encoded by Halloween genes were identified and genetically and biochemically characterized. However, the tertiary structures of these proteins have not yet been characterized. Here, we report the results of an integrated series of in silico, in vitro, and in vivo analyses of the Halloween GST protein Noppera-bo (Nobo). We determined crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with GSH and 17β-estradiol, a DmNobo inhibitor. 17β-Estradiol almost fully occupied the putative ligand-binding pocket and a prominent hydrogen bond formed between 17β-estradiol and Asp-113 of DmNobo. We found that Asp-113 is essential for 17β-estradiol–mediated inhibition of DmNobo enzymatic activity, as 17β-estradiol did not inhibit and physically interacted less with the D113A DmNobo variant. Asp-113 is highly conserved among Nobo proteins, but not among other GSTs, implying that this residue is important for endogenous Nobo function. Indeed, a homozygous nobo allele with the D113A substitution exhibited embryonic lethality and an undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue that appears to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. To the best of our knowledge, ours is the first study describing the structural characteristics of insect steroidogenic Halloween proteins. Our findings provide insights relevant for applied entomology to develop insecticides that specifically inhibit ecdysteroid biosynthesis.

scholarly journals The Essential Genome of Burkholderia cenocepacia H111

2017 ◽  
Vol 199 (22) ◽  
Author(s):  
Steven Higgins ◽  
Maria Sanchez-Contreras ◽  
Stefano Gualdi ◽  
Marta Pinto-Carbó ◽  
Aurélien Carlier ◽  
...  
Keyword(s):  
Minimal Medium ◽  
Essential Genes ◽  
Burkholderia Cenocepacia ◽  
Chromosome 1 ◽  
Biological Research ◽  
Chromosome 2 ◽  
Powerful Method ◽  
Rich Medium ◽  
Content Type ◽  
Autonomous Growth

ABSTRACT The study of the minimum set of genes required to sustain life is a fundamental question in biological research. Recent studies on bacterial essential genes suggested that between 350 and 700 genes are essential to support autonomous bacterial cell growth. Essential genes are of interest as potential new antimicrobial drug targets; hence, our aim was to identify the essential genome of the cystic fibrosis (CF) isolate Burkholderia cenocepacia H111. Using a transposon sequencing (Tn-Seq) approach, we identified essential genes required for growth in rich medium under aerobic and microoxic conditions as well as in a defined minimal medium with citrate as a sole carbon source. Our analysis suggests that 398 genes are required for autonomous growth in rich medium, a number that represents only around 5% of the predicted genes of this bacterium. Five hundred twenty-six genes were required to support growth in minimal medium, and 434 genes were essential under microoxic conditions (0.5% O2). A comparison of these data sets identified 339 genes that represent the minimal set of essential genes required for growth under all conditions tested and can be considered the core essential genome of B. cenocepacia H111. The majority of essential genes were found to be located on chromosome 1, and few such genes were located on chromosome 2, where most of them were clustered in one region. This gene cluster is fully conserved in all Burkholderia species but is present on chromosome 1 in members of the closely related genus Ralstonia, suggesting that the transfer of these essential genes to chromosome 2 in a common ancestor contributed toward the separation of the two genera. IMPORTANCE Transposon sequencing (Tn-Seq) is a powerful method used to identify genes that are essential for autonomous growth under various conditions. In this study, we have identified a set of “core essential genes” that are required for growth under multiple conditions, and these genes represent potential antimicrobial targets. We also identified genes specifically required for growth under low-oxygen and nutrient-limited environments. We generated conditional mutants to verify the results of our Tn-Seq analysis and demonstrate that one of the identified genes was not essential per se but was an artifact of the construction of the mutant library. We also present verified examples of genes that were not truly essential but, when inactivated, showed a growth defect. These examples have identified so-far-underestimated shortcomings of this powerful method.

Jun mediates Frizzled-induced R3/R4 cell fate distinction and planar polarity determination in the Drosophila eye

Development ◽  
2000 ◽  
Vol 127 (16) ◽  
pp. 3619-3629 ◽  
Author(s):  
U. Weber ◽  
N. Paricio ◽  
M. Mlodzik
Keyword(s):  
Cell Fate ◽  
Genetic Interaction ◽  
Function Analysis ◽  
Loss Of Function ◽  
Planar Polarity ◽  
Jnk Signaling ◽  
Drosophila Eye ◽  
Polarity Determination ◽  
Signaling Components

Jun acts as a signal-regulated transcription factor in many cellular decisions, ranging from stress response to proliferation control and cell fate induction. Genetic interaction studies have suggested that Jun and JNK signaling are involved in Frizzled (Fz)-mediated planar polarity generation in the Drosophila eye. However, simple loss-of-function analysis of JNK signaling components did not show comparable planar polarity defects. To address the role of Jun and JNK in Fz signaling, we have used a combination of loss- and gain-of-function studies. Like Fz, Jun affects the bias between the R3/R4 photoreceptor pair that is critical for ommatidial polarity establishment. Detailed analysis of jun(−) clones reveals defects in R3 induction and planar polarity determination, whereas gain of Jun function induces the R3 fate and associated polarity phenotypes. We find also that affecting the levels of JNK signaling by either reduction or overexpression leads to planar polarity defects. Similarly, hypomorphic allelic combinations and overexpression of the negative JNK regulator Puckered causes planar polarity eye phenotypes, establishing that JNK acts in planar polarity signaling. The observation that Dl transcription in the early R3/R4 precursor cells is deregulated by Jun or Hep/JNKK activation, reminiscent of the effects seen with Fz overexpression, suggests that Jun is one of the transcription factors that mediates the effects of fz in planar polarity generation.

scholarly journals Multiplex enCas12a screens detect functional buffering among paralogs otherwise masked in monogenic Cas9 knockout screens

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Merve Dede ◽  
Megan McLaughlin ◽  
Eiru Kim ◽  
Traver Hart
Keyword(s):  
Cell Lines ◽  
Protein Complexes ◽  
Gene Knockout ◽  
Essential Genes ◽  
Loss Of Function ◽  
Systematic Analysis ◽  
Synthetic Lethal ◽  
Synthetic Lethal Interactions ◽  
Crispr Screen ◽  
A Cell

Abstract Background Pooled library CRISPR/Cas9 knockout screening across hundreds of cell lines has identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens is low compared to the number of constitutively expressed genes in a cell. Results Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observe that half of all constitutively expressed genes are never detected in any CRISPR screen and that these never-essentials are highly enriched for paralogs. We investigated functional buffering among approximately 400 candidate paralog pairs using CRISPR/enCas12a dual-gene knockout screening in three cell lines. We observe 24 synthetic lethal paralog pairs that have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions are present in at least two out of three cell lines and 14 of 24 (58%) are present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes. Conclusions Together, these observations strongly suggest that functionally redundant paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes in monogenic CRISPR-based loss of function screens.

scholarly journals Caenorhabditis elegans as a Model Organism to Evaluate the Antioxidant Effects of Phytochemicals

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3194
Author(s):  
Begoña Ayuda-Durán ◽  
Susana González-Manzano ◽  
Ana M. González-Paramás ◽  
Celestino Santos-Buelga
Keyword(s):  
Oxidative Stress ◽  
Caenorhabditis Elegans ◽  
Model Organism ◽  
Lipid Peroxides ◽  
Biological Research ◽  
Loss Of Function ◽  
Fluorescent Reporters ◽  
C Elegans ◽  
High Degree

The nematode Caenorhabditis elegans was introduced as a model organism in biological research by Sydney Brenner in the 1970s. Since then, it has been increasingly used for investigating processes such as ageing, oxidative stress, neurodegeneration, or inflammation, for which there is a high degree of homology between C. elegans and human pathways, so that the worm offers promising possibilities to study mechanisms of action and effects of phytochemicals of foods and plants. In this paper, the genes and pathways regulating oxidative stress in C. elegans are discussed, as well as the methodological approaches used for their evaluation in the worm. In particular, the following aspects are reviewed: the use of stress assays, determination of chemical and biochemical markers (e.g., ROS, carbonylated proteins, lipid peroxides or altered DNA), influence on gene expression and the employment of mutant worm strains, either carrying loss-of-function mutations or fluorescent reporters, such as the GFP.

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