scholarly journals Characterization of a dual-active enzyme, DcpA, involved in cyclic diguanosine monophosphate turnover in Mycobacterium smegmatis

Microbiology ◽  
2014 ◽  
Vol 160 (10) ◽  
pp. 2304-2318 ◽  
Author(s):  
Indra Mani Sharma ◽  
Sunita Prakash ◽  
Thillaivillalan Dhanaraman ◽  
Dipankar Chatterji
Keyword(s):  
Mycobacterium Smegmatis ◽  
Cellular Localization ◽  
Sequence Similarity ◽  
Domain Architecture ◽  
Single Copy ◽  
Active Enzyme ◽  
Term Survival ◽  
Cyclic Diguanosine Monophosphate ◽  
Cellular Turnover ◽  
Diguanosine Monophosphate

We have reported previously that the long-term survival of Mycobacterium smegmatis is facilitated by a dual-active enzyme MSDGC-1 (renamed DcpA), which controls the cellular turnover of cyclic diguanosine monophosphate (c-di-GMP). Most mycobacterial species possess at least a single copy of a DcpA orthologue that is highly conserved in terms of sequence similarity and domain architecture. Here, we show that DcpA exists in monomeric and dimeric forms. The dimerization of DcpA is due to non-covalent interactions between two protomers that are arranged in a parallel orientation. The dimer shows both synthesis and hydrolysis activities, whereas the monomer shows only hydrolysis activity. In addition, we have shown that DcpA is associated with the cytoplasmic membrane and exhibits heterogeneous cellular localization with a predominance at the cell poles. Finally, we have also shown that DcpA is involved in the change in cell length and colony morphology of M. smegmatis. Taken together, our study provides additional evidence about the role of the bifunctional protein involved in c-di-GMP signalling in M. smegmatis.

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.

Salamander rods and cones contain distinct transducin alpha subunits

2000 ◽  
Vol 17 (6) ◽  
pp. 847-854 ◽  
Author(s):  
JAMES C. RYAN ◽  
SERGEY ZNOIKO ◽  
LIN XU ◽  
ROSALIE K. CROUCH ◽  
JIAN-XING MA
Keyword(s):  
Amino Acid ◽  
Blot Analysis ◽  
Cellular Localization ◽  
Amino Acid Level ◽  
Single Copy ◽  
Lower Vertebrate ◽  
Rods And Cones ◽  
Sequence Identity ◽  
Outer Segments ◽  
Cone Pigments

The mammalian retina is known to contain two distinct transducins that interact with their respective rod and cone pigments. However, there are no reports of a nonmammalian species having two distinct transducins. In the present study, we report the cloning and cellular localization of two transducin α subunits (Gαt) from the tiger salamander. Through degenerate polymerase chain reaction (PCR) and subsequent screening of a salamander retina cDNA library, we have identified two forms of Gαt. When compared to existing sequences in GenBank, the cloned subunits showed high similarity to rod and cone transducins. The salamander Gαt-1 has 91.2–93.7% amino acid sequence identity to mammalian rod Gαt subunits and 79.7–80.9% to mammalian cone Gαts. The salamander Gαt-2 has 86.2–87.9% sequence identity to mammalian cone Gαts and 78.9–80.9% to mammalian rod Gαts at the amino acid level. The Gαt-1 cDNA encodes 350 amino acids while the Gαt-2 cDNA encodes 354 residues, which is typical for rod and cone Gαts, respectively, and we thus identified the Gαt-1 as rod and Gαt-2 as cone Gαt. Sequences identified as effector binding sites and GTPase activity regions are highly conserved between the two subunits. Genomic Southern blot analysis showed that rod and cone Gαt subunits are both encoded by single-copy genes. Northern blot analysis identified retina-specific transcripts of 3.0 kb for rod Gαt and 2.6 kb for cone Gαt. Immunohistochemistry in the flat-mounted salamander retina demonstrated that rod Gαt is localized to rods, predominantly in the outer segments; similarly, cone Gαt is localized to cone outer segments. The results confirm that the two sequences encode rod and cone transducins and demonstrate that this lower vertebrate contains two distinct transducins that are localized specifically to rod and cone photoreceptors.

An ubiquitously expressed gene 3.5 kilobases upstream of the glycerol-3-phosphate dehydrogenase gene in mice

1989 ◽  
Vol 9 (3) ◽  
pp. 935-945
Author(s):  
L A Johnston ◽  
M A Kotarski ◽  
D J Jerry ◽  
L P Kozak
Keyword(s):  
Sequence Similarity ◽  
Single Copy ◽  
Transcriptional Unit ◽  
Chromosome 15 ◽  
Glycerol Phosphate ◽  
Reading Frame ◽  
Dehydrogenase Gene ◽  
New Gene ◽  
Or Gene ◽  
Glycerol 3 Phosphate Dehydrogenase

While studying the organization of the mouse glycerol-phosphate dehydrogenase gene (Gdc-1 on chromosome 15), we identified a novel transcriptional unit located only 3.4 kilobases (kb) upstream of the 5' end of the Gdc-1 gene. This gene has been provisionally named D15Kz1. The unusual proximity of these two genes led us to investigate the pattern of expression and sequence characteristics of the new gene for comparison with those of Gdc-1. D15Kz1 was found to have transcripts of 3.2 and 3.4 kb in length. The 3.4-kb transcript was expressed at low levels in all tissues examined, whereas the 3.2-kb transcript was detected only in the cerebral cortex and the brown fat. D15Kz1 and Gdc-1 are not coordinately regulated, as evidenced by the characteristics of their expression in several tissues and in differentiating 3T3-F442A adipocyte cultures. A cDNA sequence of 3,105 bases isolated from an embryonal carcinoma lambda gt10 cDNA library had a large open reading frame of 461 amino acids at one end followed by 1.6 kb of sequence with multiple stop codons. Algorithms used to search the protein and nucleic acid data bases detected no significant sequence similarity to any other protein or gene. Southern blot analysis of genomic DNA using the D15Kz1 cDNA as a probe indicated that D15Kz1 is a single-copy gene in the mouse genome and that it is conserved in humans, rats, and chickens. This conservation of gene sequences suggests that D15Kz1 encodes a protein with an important cellular function.

scholarly journals Proteomic and biochemical comparison of the cellular interaction partners of human VPS33A and VPS33B

2017 ◽  
Author(s):  
Morag R. Hunter ◽  
Geoffrey G. Hesketh ◽  
Anne-Claude Gingras ◽  
Stephen C. Graham
Keyword(s):  
Sequence Similarity ◽  
Domain Architecture ◽  
Gel Filtration ◽  
Cellular Interaction ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Protein Subunit ◽  
Over Expression ◽  
Biochemical Comparison

ABSTRACTMulti-subunit tethering complexes control membrane fusion events in eukaryotic cells. CORVET and HOPS are two such multi-subunit tethering complexes, both containing the Sec1/Munc18 protein subunit VPS33A. Metazoans additionally possess VPS33B, which has considerable sequence similarity to VPS33A but does not integrate into CORVET or HOPS complexes and instead stably interacts with VIPAR. It has been recently suggested that VPS33B and VIPAR comprise two subunits of a novel multi-subunit tethering complex (named ‘CHEVI’), analogous in configuration to CORVET and HOPS. We utilised the BioID proximity biotinylation assay to compare and contrast the interactomes of VPS33A and VPS33B. Overall, few proteins were identified as associating with both VPS33A and VPS33B, suggesting these proteins have distinct sub-cellular localisations. Consistent with previous reports, we observed that VPS33A was co-localised with many components of class III phosphatidylinositol 3-kinase (PI3KC3) complexes: PIK3C3, PIK3R4, NRBF2, UVRAG and RUBICON. Although in this assay VPS33A clearly co-localised with several subunits of CORVET and HOPS, no proteins with the canonical CORVET/HOPS domain architecture were found to co-localise with VPS33B. Instead, we identified two novel VPS33B-interacting proteins, VPS53 and CCDC22. CCDC22 co-immunoprecipitated with VPS33B and VIPAR in over-expression conditions and interacts directly with the VPS33B-VIPAR complex in vitro. However, CCDC22 does not appear to co-fractionate with VPS33B and VIPAR in gel filtration of human cell lysates. We also observed that the protein complex in HEK293T cells which contained VPS33B and VIPAR was considerably smaller than CORVET/HOPS, suggesting that, unlike VPS33A, VPS33B does not assemble into a large stable multi-subunit tethering complex.

scholarly journals Degradation of cyclic diguanosine monophosphate by a hybrid two-component protein protects Azoarcus sp. strain CIB from toluene toxicity

2016 ◽  
Vol 113 (46) ◽  
pp. 13174-13179 ◽  
Author(s):  
Zaira Martín-Moldes ◽  
Blas Blázquez ◽  
Claudine Baraquet ◽  
Caroline S. Harwood ◽  
María T. Zamarro ◽  
...  
Keyword(s):  
Aromatic Hydrocarbons ◽  
Response Regulator ◽  
Anaerobic Growth ◽  
Two Component System ◽  
Component System ◽  
Bacterial Physiology ◽  
Toxic Concentration ◽  
Two Component ◽  
Cyclic Diguanosine Monophosphate ◽  
Diguanosine Monophosphate

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that controls diverse functions in bacteria, including transitions from planktonic to biofilm lifestyles, virulence, motility, and cell cycle. Here we describe TolR, a hybrid two-component system (HTCS), from the β-proteobacterium Azoarcus sp. strain CIB that degrades c-di-GMP in response to aromatic hydrocarbons, including toluene. This response protects cells from toluene toxicity during anaerobic growth. Whereas wild-type cells tolerated a sudden exposure to a toxic concentration of toluene, a tolR mutant strain or a strain overexpressing a diguanylate cyclase gene lost viability upon toluene shock. TolR comprises an N-terminal aromatic hydrocarbon-sensing Per–Arnt–Sim (PAS) domain, followed by an autokinase domain, a response regulator domain, and a C-terminal c-di-GMP phosphodiesterase (PDE) domain. Autophosphorylation of TolR in response to toluene exposure initiated an intramolecular phosphotransfer to the response regulator domain that resulted in c-di-GMP degradation. The TolR protein was engineered as a functional sensor histidine kinase (TolRSK) and an independent response regulator (TolRRR). This classic two-component system (CTCS) operated less efficiently than TolR, suggesting that TolR was evolved as a HTCS to optimize signal transduction. Our results suggest that TolR enables Azoarcus sp. CIB to adapt to toxic aromatic hydrocarbons under anaerobic conditions by modulating cellular levels of c-di-GMP. This is an additional role for c-di-GMP in bacterial physiology.

scholarly journals The First Transcriptome Assembly of Yenyuan Stream Salamander (Batrachuperus yenyuanensis) Provides Novel Insights into Its Molecular Evolution

2019 ◽  
Vol 20 (7) ◽  
pp. 1529 ◽  
Author(s):  
Jianli Xiong ◽  
Yunyun Lv ◽  
Yong Huang ◽  
Qiangqiang Liu
Keyword(s):  
Sequence Similarity ◽  
Transcriptome Assembly ◽  
Repetitive Sequences ◽  
Divergence Time ◽  
Gene Families ◽  
Single Copy ◽  
Phylogenetic Position ◽  
Interesting Insight ◽  
Chinese Giant Salamander ◽  
Lineage Divergence

The Yenyuan stream salamander (Batrachuperus yenyuanensis) has been previously evaluated with regards to phylogeny, population genetics, and hematology, but genomic information is sparse due to the giant genome size of salamanders which contain highly repetitive sequences, thus resulting in the lack of a complete reference genome. This study evaluates the encoding genetic sequences and provides the first transcriptome assembly of Yenyuan stream salamander based on mixed samples from the liver, spermary, muscle and spleen tissues. Using this transcriptome assembly and available encoding sequences from other vertebrates, the gene families, phylogenetic status, and species divergence time were compared or estimated. A total of 13,750 encoding sequences were successfully obtained from the transcriptome assembly of Yenyuan stream salamander, estimated to contain 40.1% of the unigenes represented in tetrapod databases. A total of 88.79% of these genes could be annotated to a biological function by current databases. Through gene family clustering, we found multiple possible isoforms of the Scribble gene—whose function is related to regeneration—based on sequence similarity. Meanwhile, we constructed a robust phylogenetic tree based on 56 single-copy orthologues, which indicates that based on phylogenetic position, the Yenyuan stream salamander presents the closest relationship with the Chinese giant salamander (Andrias davidianus) of the investigated vertebrates. Based on the fossil-calibrated phylogeny, we estimated that the lineage divergence between the ancestral Yenyuan stream salamander and the Chinese giant salamander may have occurred during the Cretaceous period (~78.4 million years ago). In conclusion, this study not only provides a candidate gene that is valuable for exploring the remarkable capacity of regeneration in the future, but also gives an interesting insight into the understanding of Yenyuan stream salamander by this first transcriptome assembly.

scholarly journals Globins Synthesize the Second Messenger Bis-(3′–5′)-Cyclic Diguanosine Monophosphate in Bacteria

2009 ◽  
Vol 388 (2) ◽  
pp. 262-270 ◽  
Author(s):  
Xuehua Wan ◽  
Jason R. Tuckerman ◽  
Jennifer A. Saito ◽  
Tracey Allen K. Freitas ◽  
James S. Newhouse ◽  
...  
Keyword(s):  
Second Messenger ◽  
Cyclic Diguanosine Monophosphate ◽  
Diguanosine Monophosphate

scholarly journals Identification of a novel transcription unit in the human insulin-like growth factor-II gene

1991 ◽  
Vol 280 (2) ◽  
pp. 439-444 ◽  
Author(s):  
K Ikejiri ◽  
T Wasada ◽  
K Haruki ◽  
N Hizuka ◽  
Y Hirata ◽  
...  
Keyword(s):  
Growth Factor ◽  
Human Insulin ◽  
Sequence Similarity ◽  
Single Copy ◽  
Transcription Unit ◽  
Insulin Like Growth Factor ◽  
Protein Coding ◽  
Consensus Sequences ◽  
Genomic Location ◽  
Factor Ii

The human insulin-like growth factor-II (hIGF-II) gene has until now been thought to be composed of eight exons, including three independent leader exons. In the present study two additional exons, one leader exon and one alternatively used ordinate exon, have been newly identified. They were abundantly expressed in human histiocytoma tissue, generating mRNA species of about 5.0 kb in length. The new leader exon shows significant sequence similarity with the rE1 exon, previously reported to be transcribed only in the rat, and is mapped at nearly the same genomic location as in the rat. On the other hand, sequence similarity with another exon in the corresponding region of the rat genome was also found. It was, however, obvious that the rat sequence would not work as an active exon, since both splice acceptor and donor sites were deviated considerably from the consensus sequences. It has thus become apparent that the complex transcription unit of a single-copy hIGF-II gene comprises at least 10 exons, including four leader exons, one alternative exon and three common protein-coding exons.

scholarly journals Evidence for a Role of rpoE in Stressed and Unstressed Cells of Marine Vibrio angustumStrain S14

2000 ◽  
Vol 182 (24) ◽  
pp. 6964-6974 ◽  
Author(s):  
Erika Hild ◽  
Kathy Takayama ◽  
Rose-Marie Olsson ◽  
Staffan Kjelleberg
Keyword(s):  
Oxidative Stress ◽  
Blot Analysis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Sequence Similarity ◽  
Temperature Shift ◽  
Optimal Growth ◽  
Single Copy ◽  
Carbon Starvation ◽  
Wild Type

ABSTRACT We report the cloning, sequencing, and characterization of therpoE homolog in Vibrio angustum S14. TherpoE gene encodes a protein with a predicted molecular mass of 19.4 kDa and has been demonstrated to be present as a single-copy gene by Southern blot analysis. The deduced amino acid sequence of RpoE is most similar to that of the RpoE homolog of Sphingomonas aromaticivorans, ς24, displaying sequence similarity and identity of 63 and 43%, respectively. Northern blot analysis demonstrated the induction of rpoE 6, 12, and 40 min after a temperature shift to 40°C. An rpoE mutant was constructed by gene disruption. There was no difference in viability during logarithmic growth, stationary phase, or carbon starvation between the wild type and the rpoE mutant strain. In contrast, survival of the mutant was impaired following heat shock during exponential growth, as well as after oxidative stress at 24 h of carbon starvation. The mutant exhibited microcolony formation during optimal growth temperatures (22 to 30°C), and cell area measurements revealed an increase in cell volume of the mutant during growth at 30°C, compared to the wild-type strain. Moreover, outer membrane and periplasmic space protein analysis demonstrated many alterations in the protein profiles for the mutant during growth and carbon starvation, as well as following oxidative stress, in comparison with the wild-type strain. It is thereby concluded that RpoE has an extracytoplasmic function and mediates a range of specific responses in stressed as well as unstressed cells of V. angustum S14.

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