Faculty Opinions recommendation of The stability of mRNA influences the temporal order of the induction of genes encoding inflammatory molecules.

The PHYTOCHROME-INTERACTING FACTORs (PIFs) play a central role in repressing photomorphogenesis, and phosphorylation mediates the stability of PIF proteins. Although the kinases responsible for PIF phosphorylation have been extensively studied, the phosphatases that dephosphorylate PIFs remain largely unknown. Here, we report that seedlings with mutations in FyPP1 and FyPP3, 2 genes encoding the catalytic subunits of protein phosphatase 6 (PP6), exhibited short hypocotyls and opened cotyledons in the dark, which resembled the photomorphogenic development of dark-grown pifq mutants. The hypocotyls of dark-grown sextuple mutant fypp1 fypp3 (f1 f3) pifq were shorter than those of parental mutants f1 f3 and pifq, indicating that PP6 phosphatases and PIFs function synergistically to repress photomorphogenesis in the dark. We showed that FyPPs directly interacted with PIF3 and PIF4, and PIF3 and PIF4 proteins exhibited mobility shifts in f1 f3 mutants, consistent with their hyperphosphorylation. Moreover, PIF4 was more rapidly degraded in f1 f3 mutants than in wild type after light exposure. Whole-genome transcriptomic analyses indicated that PP6 and PIFs coregulated many genes, and PP6 proteins may positively regulate PIF transcriptional activity. These data suggest that PP6 phosphatases may repress photomorphogenesis by controlling the stability and transcriptional activity of PIF proteins via regulating PIF phosphorylation.

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Production of the Biocommodities Butanol and Acetone from Methanol with Fluorescent FAST-tagged Proteins using Metabolically Engineered Strains of Eubacterium Limosum

10.21203/rs.3.rs-171102/v1 ◽

2021 ◽

Author(s):

Maximilian Flaiz ◽

Gideon Ludwig ◽

Frank R. Bengelsdorf ◽

Peter Dürre

Keyword(s):

Fusion Proteins ◽

Reporter Protein ◽

Gene Encoding ◽

Fluorescent Reporter ◽

Recombinant Production ◽

Acetoacetate Decarboxylase ◽

Genes Encoding ◽

Eubacterium Limosum ◽

Fluorescent Cells ◽

The Stability

Abstract Background: The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited.Results: In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E. limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product yields during growth.Conclusions: The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E. limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.

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Molecular mechanisms and structural features of cardiomyopathy-causing troponin T mutants in the tropomyosin overlap region

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1710354114 ◽

2017 ◽

Vol 114 (42) ◽

pp. 11115-11120 ◽

Cited By ~ 13

Author(s):

Binnu Gangadharan ◽

Margaret S. Sunitha ◽

Souhrid Mukherjee ◽

Ritu Roy Chowdhury ◽

Farah Haque ◽

...

Keyword(s):

Molecular Mechanisms ◽

Troponin T ◽

Point Mutations ◽

Structural Features ◽

Sarcomeric Proteins ◽

Binding Assays ◽

Genes Encoding ◽

The Stability ◽

Insight Into

Point mutations in genes encoding sarcomeric proteins are the leading cause of inherited primary cardiomyopathies. Among them are mutations in the TNNT2 gene that encodes cardiac troponin T (TnT). These mutations are clustered in the tropomyosin (Tm) binding region of TnT, TNT1 (residues 80–180). To understand the mechanistic changes caused by pathogenic mutations in the TNT1 region, six hypertrophic cardiomyopathy (HCM) and two dilated cardiomyopathy (DCM) mutants were studied by biochemical approaches. Binding assays in the absence and presence of actin revealed changes in the affinity of some, but not all, TnT mutants for Tm relative to WT TnT. HCM mutants were hypersensitive and DCM mutants were hyposensitive to Ca2+ in regulated actomyosin ATPase activities. To gain better insight into the disease mechanism, we modeled the structure of TNT1 and its interactions with Tm. The stability predictions made by the model correlated well with the affinity changes observed in vitro of TnT mutants for Tm. The changes in Ca2+ sensitivity showed a strong correlation with the changes in binding affinity. We suggest the primary reason by which these TNNT2 mutations between residues 92 and 144 cause cardiomyopathy is by changing the affinity of TnT for Tm within the TNT1 region.

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Insulin receptor substrate 1 gene expression is strongly up-regulated by HSPB8 silencing in U87 glioma cells

Endocrine Regulations ◽

10.2478/enr-2020-0026 ◽

2020 ◽

Vol 54 (4) ◽

pp. 231-243

Author(s):

Oksana S. Hnatiuk ◽

Dariia O. Tsymbal ◽

Dmytro O. Minchenko ◽

Olena O. Khita ◽

Yulia M. Viletska ◽

...

Keyword(s):

Insulin Receptor ◽

Quantitative Polymerase Chain Reaction ◽

Glioma Cells ◽

Insulin Receptor Substrate ◽

Gene Expressions ◽

Insulin Receptor Substrate 1 ◽

Expression Of Genes ◽

Genes Encoding ◽

The Stability ◽

Related Proteins

Abstract Objective. The aim of the present investigation was to study the expression of genes encoding IRS1 (insulin receptor substrate 1) and some other functionally active proteins in U87 glioma cells under silencing of polyfunctional chaperone HSPB8 for evaluation of the possible significance of this protein in intergenic interactions. Methods. Silencing of HSPB8 mRNA was introduced by HSPB8 specific siRNA. The expression level of HSPB8, IRS1, HK2, GLO1, HOMER3, MYL9, NAMPT, PER2, PERP, GADD45A, and DEK genes was studied in U87 glioma cells by quantitative polymerase chain reaction. Results. It was shown that silencing of HSPB8 mRNA by specific to HSPB8 siRNA led to a strong down-regulation of this mRNA and significant modification of the expression of IRS1 and many other genes in glioma cells: strong up-regulated of HOMER3, GLO1, and PERP and down-regulated of MYL9, NAMPT, PER2, GADD45A, and DEK gene expressions. At the same time, no significant changes were detected in the expression of HK2 gene in glioma cells treated by siRNA, specific to HSPB8. Moreover, the silencing of HSPB8 mRNA enhanced the glioma cells proliferation rate. Conclusions. Results of this investigation demonstrated that silencing of HSPB8 mRNA affected the expression of IRS1 gene as well as many other genes encoding tumor growth related proteins. It is possible that the dysregulation of most of the studied genes in glioma cells after silencing of HSPB8 is reflected by a complex of intergenic interactions and that this polyfunctional chaperone is an important factor for the stability of genome function and regulatory mechanisms contributing to the tumorigenesis control.

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The role of the yolk syncytial layer in germ layer patterning in zebrafish

Development ◽

10.1242/dev.127.21.4681 ◽

2000 ◽

Vol 127 (21) ◽

pp. 4681-4689 ◽

Cited By ~ 5

Author(s):

S. Chen ◽

D. Kimelman

Keyword(s):

Related Factor ◽

Homeodomain Protein ◽

Germ Layer ◽

Cell Stage ◽

Yolk Syncytial Layer ◽

Genes Encoding ◽

The Stability

Formation of the three germ layers requires a series of inductive events during early embryogenesis. Studies in zebrafish indicate that the source of these inductive signals may be the extra-embryonic yolk syncytial layer (YSL). The characterization of genes encoding the nodal-related factor, Squint, and homeodomain protein, Bozozok, both of which are expressed in the YSL, suggested that the YSL has a role in mesendoderm induction. However, these genes, and a second nodal-related factor, cyclops, are also expressed in the overlying marginal blastomeres, raising the possibility that the marginal blastomeres can induce mesendodermal genes independently of the YSL. We have developed a novel technique to study signaling from the YSL in which we specifically eliminate RNAs in the YSL, thus addressing the in vivo requirement of RNA-derived signals from this region in mesendoderm induction. We show that injection of RNase into the yolk cell after the 1K cell stage (3 hours) effectively eliminates YSL transcripts without affecting ubiquitously expressed genes in the blastoderm. We also present data that indicate the stability of existing proteins in the YSL is unaffected by RNase injection. Using this technique, we show that RNA in the YSL is required for the formation of ventrolateral mesendoderm and induction of the nodal-related genes in the ventrolateral marginal blastomeres, revealing the presence of an unidentified inducing signal released from the YSL. We also demonstrate that the dorsal mesoderm can be induced independently of signals from the YSL and present evidence that this is due to the stabilization of (β)-catenin in the dorsal marginal blastomeres. Our results demonstrate that germ layer formation and patterning in zebrafish uses a combination of YSL-dependent and -independent inductive events.

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Comparative genomics in “Candidatus Kuenenia stuttgartiensis” reveal high genomic plasticity in the overall genome structure, CRISPR loci and surface proteins

BMC Genomics ◽

10.1186/s12864-020-07242-1 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Chang Ding ◽

Lorenz Adrian

Keyword(s):

Growth Rate ◽

Genome Structure ◽

Draft Genome ◽

Surface Proteins ◽

Anammox Bacteria ◽

Continuous Stirred Tank Reactor ◽

Genes Encoding ◽

Continuous Stirred Tank ◽

The Stability ◽

Genomic Regions

Abstract Background Anaerobic ammonium oxidizing bacteria (anammox bacteria) are contributing significantly to the nitrogen cycle and are successfully used in wastewater treatment. Due to the lack of complete genomes in the databases, little is known about the stability and variability of their genomes and how the genomes evolve in response to changing environments. Results Here we report the complete genome of the anammox bacterium “Candidatus Kuenenia stuttgartiensis” strain CSTR1 which was enriched planktonically in a semi-continuous stirred-tank reactor. A comparison of the genome of strain CSTR1 with the genome of “Ca. Kuenenia stuttgartiensis” MBR1 and the draft genome of KUST showed > 99% average nucleotide identity among all. Rearrangements of large genomic regions were observed, most of which were associated with transposase genes. Phylogenetic analysis suggests that strain MBR1 is more distantly related to the other two strains. Proteomic analysis of actively growing cells of strain CSTR1 (growth rate ~ 0.33 d− 1) failed to detect the annotated cytochrome cd1-type nitrite reductase (NirS) although in total 1189 proteins were found in the proteome. Yet, this NirS was expressed when strain CSTR1 was under stress or starvation (growth rate < 0.06 d− 1). We also observed large sequence shifts in the strongly expressed S-layer protein compared to other “Ca. Kuenenia” strains, indicating the formation of hybrids of genes encoding the surface proteins. Conclusions “Ca. Kuenenia” strains appear to be relatively stable in their basic physiological traits, but show high variability in overall genome structure and surface proteins.

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