Rag A/Gtr1p are G proteins and are known to be involved in the RCC1-Ran pathway. We employed the two-hybrid method using Rag A as the bait to identify proteins binding to Rag A, and we isolated two novel human G proteins, Rag C and Rag D. Rag C demonstrates homology with Rag D (81.1% identity) and with Gtr2p of Saccharomyces cerevisiae (46.1% identity), and it belongs to the Rag A subfamily of the Ras family. Rag C and Rag D contain conserved GTP-binding motifs (PM-1, -2, and -3) in their N-terminal regions. Recombinant glutathione S-transferase fusion protein of Rag C efficiently bound to both [ 3 H]GTP and [ 3 H]GDP. Rag A was associated with both Rag C and Rag D in their C-terminal regions where a potential leucine zipper motif and a coiled-coil structure were found. Rag C and D were associated with both the GDP and GTP forms of Rag A. Both Rag C and Rag D changed their subcellular localization, depending on the nucleotidebound state of Rag A. In a similar way, the disruption of S. cerevisiae GTR1 resulted in a change in the localization of Gtr2p.G proteins are a superfamily of regulatory GTP hydrolases and are composed of a large number of proteins. These include Ras family proteins, hetrotrimeric G protein ␣ subunits, and elongation factors TU and G, among others (1). Ras-like small G proteins such as Ras, Rab, Rho, ARF, and Ran are monomeric and bind to the guanine nucleotides, GTP or GDP, to function as molecular switches while also playing crucial roles in cell growth, differentiation, and protein traffic between different compartments within the cells (2, 3). Ras is a key regulator of cell growth and is an essential component of the signal transduction pathways initiated by receptor tyrosine kinase (4). The Rho family members consist of Rho, Rac, and Cdc42 subtypes that control the actin cytoskeleton and that play a role in the regulation of transcription (5). ADP-ribosylation factors play a role in the vesicular trafficking pathway (6). The Rab subfamily plays a role in secretory and endocytic pathways and is located within a distinct cellular compartment (7).Ran is a well characterized nuclear Ras-like small G protein that plays an essential role in the import and export of proteins and RNAs across the nuclear membrane through the nuclear pore complex (8) and also plays a role in the induction of microtubule self-organization in Xenopus egg extracts (9 -14). There are a large number of factors that interact with either the GDP-bound form or the GTP-bound form of Ran (Gsp1p), these being nucleoporin, RanBP2/NUP358 (15, 16), Prp20p interacting protein, RanBP3(Yrb2p) (17-19), the exosome involved in ribosomal RNA processing, Dis3p (20, 21), microtubule nucleation, RanBPM (22), and regulators of Ran, Ran-GAP1 (23-25), RanBP1(Yrb1p) (26, 27), RCC1/RanGEF (28), and Mog1p (29).RCC1 catalyzes guanine nucleotide exchange on Ran (30) and is located inside the nucleus, bound to chromatin (31). The concentration of GTP within the cell is ϳ30 times higher than the concentration of GDP, thus resulting in the prefer...
BackgroundRed cell distribution width (RDW), one of many routinely examined parameters, shows the heterogeneity in erythrocyte size. We investigated the association of RDW levels with clinical parameters and prognosis of lung cancer patients.MethodsClinical and laboratory data from 332 patients with lung cancer in a single institution were retrospectively studied by univariate analysis. Kaplan-Meier survival analysis and Cox proportional hazard models were used to examine the effect of RDW on survival.ResultsThe RDW levels were divided into two groups: high RDW (>=15%), n=73 vs. low RDW, n=259 (<15%). Univariate analysis showed that there were significant associations of high RDW values with cancer stage, performance status, presence of other disease, white blood cell count, hemoglobin, mean corpuscular volume, platelet count, albumin level, C-reactive protein level, and cytokeratin 19 fragment level. Kruskal-Wallis tests revealed an association of RDW values with cancer stage in patients irrespective of comorbidity (patient with/without comorbidity: p<0.0001, patient without comorbidity: p<0.0001). Stages I-IV lung cancer patients with higher RDW values had poorer prognoses than those with lower RDW values (Wilcoxon test: p=0.002). In particular, the survival rates of stage I and II patients (n=141) were lower in the high RDW group (n=19) than in the low RDW group (n=122) (Wilcoxon test: p<0.001). Moreover, multivariate analysis showed higher RDW is a significant prognostic factor (p=0.040).ConclusionRDW is associated with several factors that reflect inflammation and malnutrition in lung cancer patients. Moreover, high levels of RDW are associated with poor survival. RDW might be used as a new and convenient marker to determine a patient’s general condition and to predict the mortality risk of lung cancer patients.
We isolated, sequenced, and characterized the cryptic plasmid pRE8424 from Rhodococcus erythropolis DSM8424. Plasmid pRE8424 is a 5,987-bp circular plasmid; it carries six open reading frames and also contains cis-acting elements, specifically a single-stranded origin and a double-stranded origin, which are characteristic of rolling-circle-replication plasmids. Experiments with pRE8424 derivatives carrying a mutated single-stranded origin sequence showed that single-stranded DNA intermediates accumulated in the cells because of inefficient conversion from single-stranded DNA to double-stranded DNA. This result indicates that pRE8424 belongs to the pIJ101/pJV1 family of rolling-circle-replication plasmids. Expression vectors that are functional in several Rhodococcus species were constructed by use of the replication origin from pRE8424. We previously reported a cryptic plasmid, pRE2895, from R. erythropolis, which may replicate by a -type mechanism, like ColE2 plasmids. The new expression vectors originating from pRE8424 were compatible with those derived from pRE2895. Coexpression experiments with these compatible expression vectors indicated that the plasmids are suitable for the simultaneous expression of multiple recombinant proteins.Rhodococcus erythropolis is a gram-positive, high-GϩC-content bacterium (Actinobacteria) that can grow at temperatures ranging from 4 to 35°C (39). Because many Rhodococcus strains have broad metabolic diversity and an array of unique enzymatic capabilities, they are of interest for pharmaceutical, environmental, chemical, and energy studies as well as for applications in the desulfurization of fossil fuels (25) and the industrial production of acrylamide (21). Several researchers have developed various genetic tools to analyze Rhodococcus (9), including Escherichia coli-Rhodococcus shuttle vectors (5) and a gene disruption system (38).We have developed inducible expression vectors (pTip vectors) that work in several Rhodococcus species (26). The pTip vectors are tightly regulated expression vectors containing a tipA promoter (P tipA ) (4, 14), from which protein expression is induced by the antibiotic reagent thiostrepton (26). In our previous report, we showed that pTip vectors mediate heterologous and homologous protein expression, as they contain multiple cloning sites for 11 restriction enzyme sites and a hexahistidine (six-His) tag sequence and they work over a wide temperature range, from 4 to 35°C. The expression yields of recombinant proteins can be up to 10 mg per liter of R. erythropolis culture. In addition, some proteins that could not be expressed in E. coli were successfully expressed in R. erythropolis.The pTip vectors carry the repAB operon of the cryptic plasmid pRE2895, which is necessary for the autonomous replication of the plasmids in Rhodococcus cells. The repAB operon encodes the replication proteins RepA and RepB, which are characteristic of pAL5000-type plasmids (26,35). The replication mechanisms of pAL5000 and pRE2895 are unknown, but RepA proteins of...
Reliable methods for conditional gene silencing in bacteria have been elusive. To improve silencing by expressed antisense RNAs (asRNAs), we systematically altered several design parameters and targeted multiple reporter and essential genes in Escherichia coli. A paired termini (PT) design, where flanking inverted repeats create paired dsRNA termini, proved effective. PTasRNAs targeted against the ackA gene within the acetate kinase-phosphotransacetylase operon (ackA-pta) triggered target mRNA decay and a 78% reduction in AckA activity with high genetic penetrance. PTasRNAs are abundant and stable and function through an RNase III independent mechanism that requires a large stoichiometric excess of asRNA. Conditional ackA silencing reduced carbon flux to acetate and increased heterologous gene expression. The PT design also improved silencing of the essential fabI gene. Full anti-fabI PTasRNA induction prevented growth and partial induction sensitized cells to a FabI inhibitor. PTasRNAs have potential for functional genomics, antimicrobial discovery and metabolic flux control.
BackgroundGenes essential for bacterial growth are of particular scientific interest. Many putative essential genes have been identified or predicted in several species, however, little is known about gene expression requirement stringency, which may be an important aspect of bacterial physiology and likely a determining factor in drug target development.Methodology/Principal FindingsWorking from the premise that essential genes differ in absolute requirement for growth, we describe silencing of putative essential genes in E. coli to obtain a titration of declining growth rates and transcript levels by using antisense peptide nucleic acids (PNA) and expressed antisense RNA. The relationship between mRNA decline and growth rate decline reflects the degree of essentiality, or stringency, of an essential gene, which is here defined by the minimum transcript level for a 50% reduction in growth rate (MTL50). When applied to four growth essential genes, both RNA silencing methods resulted in MTL50 values that reveal acpP as the most stringently required of the four genes examined, with ftsZ the next most stringently required. The established antibacterial targets murA and fabI were less stringently required.ConclusionsRNA silencing can reveal stringent requirements for gene expression with respect to growth. This method may be used to validate existing essential genes and to quantify drug target requirement.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.