scholarly journals The Plant Specific Insert as an Unconventional Signal in the Route to Plant Vacuoles

2019 ◽  
Author(s):  
Vanessa Vieira ◽  
Bruno Peixoto ◽  
Mónica Costa ◽  
Susana Pereira ◽  
José Pissarra ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Site Directed Mutagenesis ◽  
Research Gaps ◽  
Glycosylation Pattern ◽  
High Sequence Similarity ◽  
Post Translational Modifications ◽  
Plant Vacuoles ◽  
Prevacuolar Compartment ◽  
Cardosin A ◽  
Comprehensive Study

In plant cells the conventional route to the vacuole involves the endoplasmic reticulum, the Golgi and the prevacuolar compartment. However, over the years, unconventional sorting to the vacuole, bypassing the Golgi, has been described, which is the case of the Plant Specific Insert (PSI) of the aspartic proteinase cardosin A. Interestingly, this Golgi-bypass ability is not a characteristic shared by all PSIs, since two related PSIs showed to have different sensitivity to ER-to-Golgi blockage. Given the high sequence similarity between the PSIs domains, we sought to depict the differences in terms of post-translational modifications. In fact, one feature that draws our attention is that one is N-glycosylated and the other one is not. Using site-directed mutagenesis to obtain mutated versions of the two PSIs, with and without the glycosylation motif, we observed that altering the glycosylation pattern interferes with the trafficking of the protein as the non-glycosylated PSI-B, unlike its native glycosylated form, is able to bypass ER-to-Golgi blockage and accumulate in the vacuole. This is also true when the PSI domain is analyzed in the context of the full-length cardosin. Regardless of opening exciting research gaps, the results obtained so far need a more comprehensive study of the mechanisms behind this unconventional direct sorting to the vacuole.

scholarly journals N-Linked Glycosylation Modulates Golgi-Independent Vacuolar Sorting Mediated by the Plant Specific Insert

Plants ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 312 ◽  
Author(s):  
Vanessa Vieira ◽  
Bruno Peixoto ◽  
Mónica Costa ◽  
Susana Pereira ◽  
José Pissarra ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Site Directed Mutagenesis ◽  
Research Gaps ◽  
Glycosylation Pattern ◽  
High Sequence Similarity ◽  
Post Translational Modifications ◽  
Vacuolar Sorting ◽  
Prevacuolar Compartment ◽  
Cardosin A ◽  
Comprehensive Study

In plant cells, the conventional route to the vacuole involves the endoplasmic reticulum, the Golgi and the prevacuolar compartment. However, over the years, unconventional sorting to the vacuole, bypassing the Golgi, has been described, which is the case of the Plant-Specific Insert (PSI) of the aspartic proteinase cardosin A. Interestingly, this Golgi-bypass ability is not a characteristic shared by all PSIs, since two related PSIs showed to have different sensitivity to ER-to-Golgi blockage. Given the high sequence similarity between the PSI domains, we sought to depict the differences in terms of post-translational modifications. In fact, one feature that draws our attention is that one is N-glycosylated and the other one is not. Using site-directed mutagenesis to obtain mutated versions of the two PSIs, with and without the glycosylation motif, we observed that altering the glycosylation pattern interferes with the trafficking of the protein as the non-glycosylated PSI-B, unlike its native glycosylated form, is able to bypass ER-to-Golgi blockage and accumulate in the vacuole. This is also true when the PSI domain is analyzed in the context of the full-length cardosin. Regardless of opening exciting research gaps, the results obtained so far need a more comprehensive study of the mechanisms behind this unconventional direct sorting to the vacuole.

scholarly journals Baculovirus expression of the 11 mycoreovirus-1 genome segments and identification of the guanylyltransferase-encoding segment

2007 ◽  
Vol 88 (1) ◽  
pp. 342-350 ◽  
Author(s):  
S. Supyani ◽  
Bradley I. Hillman ◽  
Nobuhiro Suzuki
Keyword(s):  
Active Site ◽  
Sequence Similarity ◽  
Expression System ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Sequence Alignments ◽  
High Sequence Similarity ◽  
Chestnut Blight Fungus ◽  
A Genome ◽  
Hypovirulent Strain

The type member Mycoreovirus 1 (MyRV-1) of a newly described genus, Mycoreovirus, isolated from a hypovirulent strain 9B21 of the chestnut blight fungus, has a genome composed of 11 dsRNA segments (S1–S11). All of the segments have single ORFs on their capped, positive-sense strands. Infection of insect cells by baculovirus recombinants carrying full-length cDNAs of S1–S11 resulted in overexpression of protein products of the expected sizes, based on their deduced amino acid sequences. This expression system was utilized to identify the S3-encoded protein (VP3) as a guanylyltransferase by an autoguanylylation assay, in which only VP3 was radiolabelled with [α-32P]GTP. A series of progressive N-terminal and C-terminal deletion mutants was made to localize the autoguanylylation-active site of VP3 to aa residues 133–667. Within this region, a sequence stretch (aa 170–250) with relatively high sequence similarity to homologues of two other mycoreoviruses and two coltiviruses was identified. Site-directed mutagenesis of conserved aa residues revealed that H233, H242, Y243, F244 and F246, but not K172 or K202, play critical roles in guanylyltransferase activities. Together with broader sequence alignments of ‘turreted’ reoviruses, these results supported the a/vxxHx8Hyf/lvf motif, originally noted for orthoreovirus and aquareoviruses, as an active site for guanylyltransferases of viruses within the Orthoreovirus, Aquareovirus, Cypovirus, Oryzavirus, Fijivirus, Coltivirus and Mycoreovirus genera, as well as for the proposed Dinovernavirus genus.

scholarly journals Biosynthesis of the Tricyclic Aromatic Type II Polyketide Rishirilide: New Potential Third Ring Oxygenation after Three Cyclization Steps

2021 ◽  
Author(s):  
Ahmad Alali ◽  
Lin Zhang ◽  
Jianyu Li ◽  
Chijian Zuo ◽  
Dimah Wassouf ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Flavin Reductase ◽  
High Similarity ◽  
Dynamic Simulations ◽  
High Sequence Similarity ◽  
The Past ◽  
Biosynthetic Studies

AbstractRishirilides are a group of PKS II secondary metabolites produced by Streptomyces bottropensis Gö C4/4. Biosynthetic studies in the past have elucidated early and late steps of rishirilide biosynthesis. This work is aiming to solve the remaining steps in the rishirilide biosynthesis. Inactivation of the cyclase gene rslC3 in Streptomyces bottropensis resulted in an interruption of rishirilide production. Instead, accumulation of the tricyclic aromatic galvaquinones was observed. Similar results were observed after deletion of rslO4. Closer inspection into RslO4 crystal structure in addition to site-directed mutagenesis and molecular dynamic simulations revealed that RslO4 might be responsible for quinone formation on the third ring. The RslO1 three-dimensional structure shows a high similarity to FMN-dependent luciferase-like monooxygenases such as the epoxy-forming MsnO8 which acts with the flavin reductase MsnO3 in mensacarcin biosynthesis in the same strain. The high sequence similarity between RslO2 and MsnO3 suggests that RslO2 provides RslO1 with reduced FMN to form an epoxide that serves as substrate for RslO5.

scholarly journals Different Roles of DosS and DosT in the Hypoxic Adaptation of Mycobacteria

2010 ◽  
Vol 192 (19) ◽  
pp. 4868-4875 ◽  
Author(s):  
Min-Ju Kim ◽  
Kwang-Jin Park ◽  
In-Jeong Ko ◽  
Young Min Kim ◽  
Jeong-Il Oh
Keyword(s):  
Oxygen Tension ◽  
Response Regulator ◽  
Sequence Similarity ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
High Sequence Similarity ◽  
Hypoxic Adaptation ◽  
The Difference ◽  
A Domains

ABSTRACT The DosS (DevS) and DosT histidine kinases form a two-component system together with the DosR (DevR) response regulator in Mycobacterium tuberculosis. DosS and DosT, which have high sequence similarity to each other over the length of their amino acid sequences, contain two GAF domains (GAF-A and GAF-B) in their N-terminal sensory domains. Complementation tests in conjunction with phylogenetic analysis showed that DevS of Mycobacterium smegmatis is more closely related to DosT than DosS. We also demonstrated in vivo that DosS and DosT of M. tuberculosis play a differential role in hypoxic adaptation. DosT responds to a decrease in oxygen tension more sensitively and strongly than DosS, which might be attributable to their different autooxidation rates. The different responsiveness of DosS and DosT to hypoxia is due to the difference in their GAF-A domains accommodating the hemes. Multiple alignment analysis of the GAF-A domains of mycobacterial DosS (DosT) homologs and subsequent site-directed mutagenesis revealed that just one substitution of E87, D90, H97, L118, or T169 of DosS with the corresponding residue of DosT is sufficient to convert DosS to DosT with regard to the responsiveness to changes in oxygen tension.

Identification of active-site residues in Bradyrhizobium japonicum malonamidase E2

2000 ◽  
Vol 349 (2) ◽  
pp. 501-507 ◽  
Author(s):  
Hyun Min KOO ◽  
Sung-Ook CHOI ◽  
Hyun Mi KIM ◽  
Yu Sam KIM
Keyword(s):  
Bradyrhizobium Japonicum ◽  
Catalytic Mechanism ◽  
Sequence Similarity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Basic Residue ◽  
High Sequence Similarity ◽  
Signature Sequences ◽  
The Common

Malonamidase (MA) E2 was previously purified and characterized from Bradyrhizobium japonicum USDA 110. The gene encoding this enzyme has been cloned, sequenced and expressed in Escherichia coli. The recombinant MAE2 was purified to homogeneity from the transformed E. coli. The biochemical properties of the recombinant enzyme are essentially identical to those from wild-type B. japonicum. A database search showed that the MAE2 protein has a high sequence similarity with the common signature sequences of the amidase family. The only exception is that the aspartic residue in these signature sequences is replaced by a glutamine residue. In order to identify amino acid residues essential for enzyme activity, a series of site-directed mutagenesis studies and steady-state kinetic experiments were performed. Gln195, Ser199, Cys207 and Lys213 of the common signature sequences were selected for site-directed mutagenesis. Among the mutants, Q195D, Q195E and S199C showed less than 0.02% of the kcat value of the wild-type enzyme, and S199A, Q195L and Q195N exhibited no detectable catalytic activities. Mutants (K213L, K213R and K213H) obtained by replacement of the only conserved basic residue, Lys213, in the signature sequences, also displayed significant reductions (approx. 380-fold) in kcat value, whereas C207A kept full activity. These results suggest that MAE2 may catalyse hydrolysis of malonamate by a novel catalytic mechanism, in which Gln195, Ser199 and Lys213 are involved.

scholarly journals Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

2001 ◽  
Vol 67 (2) ◽  
pp. 673-679 ◽  
Author(s):  
Kazuhiko Ishikawa ◽  
Hiroyasu Ishida ◽  
Ikuo Matsui ◽  
Yutaka Kawarabayasi ◽  
Hisasi Kikuchi
Keyword(s):  
Active Site ◽  
Sequence Similarity ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Vector System ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
High Sequence Similarity ◽  
Pyrococcus Horikoshii ◽  
Thermophilic Archaeon

ABSTRACT Genome sequencing of the thermophilic archaeon Pyrococcus horikoshii OT3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase ofSulfolobus solfataricus and also to that encoding the aminoacylase from Bacillus stearothermophilus. The gene from P. horikoshii comprises an open reading frame of 1,164 bp with an ATG initiation codon and a TGA termination codon, encoding a 43,058-Da protein of 387 amino acid residues. However, some of the proposed active-site residues for carboxypeptidase were not found in this gene. The gene was overexpressed in Escherichia coli with the pET vector system, and the expressed enzyme had high hydrolytic activity for both carboxypeptidase and aminoacylase at high temperatures. The enzyme was stable at 90°C, with the highest activity above 95°C. The enzyme contained one bound zinc ion per one molecule that was essential for the activity. The results of site-directed mutagenesis of Glu367, which corresponds to the essential Glu270 in bovine carboxypeptidase A and the essential Glu in other known carboxypeptidases, revealed that Glu367 was not essential for this enzyme. The results of chemical modification of the SH group and site-directed mutagenesis of Cys102 indicated that Cys102 was located at the active site and was related to the activity. From these findings, it was proven that this enzyme is a hyperthermostable, bifunctional, new zinc-dependent metalloenzyme which is structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase. Some characteristics of this enzyme suggested that carboxypeptidase and aminoacylase might have evolved from a common origin.

scholarly journals Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Mohamed Ramadan ◽  
Muna Alariqi ◽  
Yizan Ma ◽  
Yanlong Li ◽  
Zhenping Liu ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Sequence Similarity ◽  
High Specificity ◽  
Transformation Method ◽  
High Sequence Similarity ◽  
Single Experiment ◽  
Next Generation Sequencing Technology ◽  
Cotton Transformation ◽  
Assembly Method ◽  
Multiple Genes

Abstract Background Upland cotton (Gossypium hirsutum), harboring a complex allotetraploid genome, consists of A and D sub-genomes. Every gene has multiple copies with high sequence similarity that makes genetic, genomic and functional analyses extremely challenging. The recent accessibility of CRISPR/Cas9 tool provides the ability to modify targeted locus efficiently in various complicated plant genomes. However, current cotton transformation method targeting one gene requires a complicated, long and laborious regeneration process. Hence, optimizing strategy that targeting multiple genes is of great value in cotton functional genomics and genetic engineering. Results To target multiple genes in a single experiment, 112 plant development-related genes were knocked out via optimized CRISPR/Cas9 system. We optimized the key steps of pooled sgRNAs assembly method by which 116 sgRNAs pooled together into 4 groups (each group consisted of 29 sgRNAs). Each group of sgRNAs was compiled in one PCR reaction which subsequently went through one round of vector construction, transformation, sgRNAs identification and also one round of genetic transformation. Through the genetic transformation mediated Agrobacterium, we successfully generated more than 800 plants. For mutants identification, Next Generation Sequencing technology has been used and results showed that all generated plants were positive and all targeted genes were covered. Interestingly, among all the transgenic plants, 85% harbored a single sgRNA insertion, 9% two insertions, 3% three different sgRNAs insertions, 2.5% mutated sgRNAs. These plants with different targeted sgRNAs exhibited numerous combinations of phenotypes in plant flowering tissues. Conclusion All targeted genes were successfully edited with high specificity. Our pooled sgRNAs assembly offers a simple, fast and efficient method/strategy to target multiple genes in one time and surely accelerated the study of genes function in cotton.

scholarly journals Evolution of Chi motifs in Proteobacteria

2021 ◽  
Author(s):  
Angélique Buton ◽  
Louis-Marie Bobay
Keyword(s):  
Sequence Similarity ◽  
Bacterial Species ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Large Dataset ◽  
High Sequence Similarity ◽  
Strand Breaks ◽  
E Coli ◽  
Dna Motif ◽  
And Staphylococcus Aureus

Abstract Homologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex allows bacteria to repair DNA double strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized in Escherichia coli and analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species: E. coli, Haemophilus influenzae, Bacillus subtilis, Lactococcus lactis and Staphylococcus aureus. In this study we detected putative Chi motifs in a large dataset of Proteobacteria and we identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif of E. coli in 85 species of Proteobacteria. Most Chi motifs were detected in Enterobacteriaceae and this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria.

Meiotic Recombination Between Paralogous RBCSB Genes on Sister Chromatids of Arabidopsis thaliana

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 947-957 ◽  
Author(s):  
John G Jelesko ◽  
Kristy Carter ◽  
Whitney Thompson ◽  
Yuki Kinoshita ◽  
Wilhelm Gruissem
Keyword(s):  
Gene Cluster ◽  
Dna Sequences ◽  
Meiotic Recombination ◽  
Sequence Similarity ◽  
Specific Gene ◽  
High Sequence Similarity ◽  
Paralogous Genes ◽  
Chimeric Genes ◽  
Unequal Recombination ◽  
Sister Chromatids

Abstract Paralogous genes organized as a gene cluster can rapidly evolve by recombination between misaligned paralogs during meiosis, leading to duplications, deletions, and novel chimeric genes. To model unequal recombination within a specific gene cluster, we utilized a synthetic RBCSB gene cluster to isolate recombinant chimeric genes resulting from meiotic recombination between paralogous genes on sister chromatids. Several F1 populations hemizygous for the synthRBCSB1 gene cluster gave rise to Luc+ F2 plants at frequencies ranging from 1 to 3 × 10-6. A nonuniform distribution of recombination resolution sites resulted in the biased formation of recombinant RBCS3B/1B::LUC genes with nonchimeric exons. The positioning of approximately half of the mapped resolution sites was effectively modeled by the fractional length of identical DNA sequences. In contrast, the other mapped resolution sites fit an alternative model in which recombination resolution was stimulated by an abrupt transition from a region of relatively high sequence similarity to a region of low sequence similarity. Thus, unequal recombination between paralogous RBCSB genes on sister chromatids created an allelic series of novel chimeric genes that effectively resulted in the diversification rather than the homogenization of the synthRBCSB1 gene cluster.

scholarly journals The Complete Chloroplast Genome of the Vulnerable Oreocharis esquirolii (Gesneriaceae): Structural Features, Comparative and Phylogenetic Analysis

Plants ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1692
Author(s):  
Li Gu ◽  
Ting Su ◽  
Ming-Tai An ◽  
Guo-Xiong Hu
Keyword(s):  
Phylogenetic Analysis ◽  
Sequence Similarity ◽  
Single Copy ◽  
Structural Features ◽  
Rrna Genes ◽  
Trna Genes ◽  
Sequencing Data ◽  
High Sequence Similarity ◽  
Plastid Genomes ◽  
Cp Genome

Oreocharis esquirolii, a member of Gesneriaceae, is known as Thamnocharis esquirolii, which has been regarded a synonym of the former. The species is endemic to Guizhou, southwestern China, and is evaluated as vulnerable (VU) under the International Union for Conservation of Nature (IUCN) criteria. Until now, the sequence and genome information of O. esquirolii remains unknown. In this study, we assembled and characterized the complete chloroplast (cp) genome of O. esquirolii using Illumina sequencing data for the first time. The total length of the cp genome was 154,069 bp with a typical quadripartite structure consisting of a pair of inverted repeats (IRs) of 25,392 bp separated by a large single copy region (LSC) of 85,156 bp and a small single copy region (SSC) of18,129 bp. The genome comprised 114 unique genes with 80 protein-coding genes, 30 tRNA genes, and four rRNA genes. Thirty-one repeat sequences and 74 simple sequence repeats (SSRs) were identified. Genome alignment across five plastid genomes of Gesneriaceae indicated a high sequence similarity. Four highly variable sites (rps16-trnQ, trnS-trnG, ndhF-rpl32, and ycf 1) were identified. Phylogenetic analysis indicated that O. esquirolii grouped together with O. mileensis, supporting resurrection of the name Oreocharis esquirolii from Thamnocharisesquirolii. The complete cp genome sequence will contribute to further studies in molecular identification, genetic diversity, and phylogeny.

Sign in / Sign up

Export Citation Format

Share Document