Differential mRNA stability controls relative gene expression within the plasmid-encoded arsenical resistance operon.

The arsenical resistance (ars) operon of the conjugative plasmid R773 encodes an ATP-driven anion extrusion pump, conferring bacterial resistance to arsenicals. The operon contains a regulatory gene, arsR, and three structural genes, arsA, arsB, and arsC. The hydrophilic ArsA and ArsC proteins are produced in large amounts, but the hydrophobic ArsB protein, an integral membrane polypeptide, is synthesized in limited quantities. Northern (RNA-DNA) hybridizations provide evidence that the inducible operon is regulated at the level of transcription. The genes were transcribed in the presence of an inducer (arsenite) as a single polycistronic mRNA with an approximate size of 4.4 kilobases (kb). This transcript was processed to generate relatively stable mRNA species: one of 2.7 kb, encoding the ArsR and ArsA proteins, and a second of 0.5 kb, encoding the ArsC protein. Segmental differences in stability within the polycistronic transcript are proposed to account for the differential expression of the ars genes. In addition, analysis of the mRNA structure at the 5' end of arsB suggests a potential translational block to the synthesis of this membrane protein

Construction of a Chimeric ArsA-ArsB Protein for Overexpression of the Oxyanion-translocating ATPase*

Resistance to toxic oxyanions of arsenic and antimony in Escherichia coli is conferred by the conjugative R-factor R773, which encodesa n ATP-driven anion extrusion pump. The ars operon is composed of three structuralg enes, arsA, arsBa, nd arsC. Although transcribed as a single unit, the three genes are differentially expressed as a result of translational differences, such that the ArsA and ArsC proteins are produced in high amounts relative to the amounot f ArsB protein made. Consequently, biochemical characterization of the ArsB protein, which is an integrmale mbrane protein containing the anion-conducting pathway, has been limited, precluding studies of the mechanism of this oxyanion pump. To overexpress tahres B gene, a series of changes were made. First, the second codon, an infrequently used leucine codon, was changed to a more frequently utilized codon. Second, a GC-rich stem-loop (AG = -17 kcal/mol) between the third and twelftcho dons was destabilized by changing several of the bases of the base-paired region. Third, the re-engineered arsB gene was fused 3’ in frame to the first 1458b ase pairs of the arsA gene to encodea 914-residue chimeric protein (486 residouf eths e ArsA protein plus 428 residues of the mutated ArsB protein) containing the entire re-engineered ArsB sequence except for the initiatinmg ethionine. The ArsA-ArsB chimera has been overexpressed at -15-20% of the total membrane proteins. Cells producing the chimeric ArsA-ArsB protein with an arsA gene in trans excluded 73AsO; from cells, demonstrating that the chimera can function as a component of the oxyaniontranslocating ATPase

H. Gregg Lewis Memorial Comments

Economic

Anharmonic effects in the A15 compounds induced by sublattice distortions

We demonstrate that elastic anomalies and lattice instabilities in the the A15 compounds are describable in terms of first-principles LDA electronic structure calculations. We show that at T=0 V_3Si, V_3Ge, and Nb_3Sn are intrinsically unstable against shears with elastic moduli C_11-C_12 and C_44, and that the zone center phonons, Gamma_2 and Gamma_12, are either unstable or extremely soft. We demonstrate that sublattice relaxation (internal strain) effects are key to understanding the behavior of the A15 materials.Comment: 5 pages, RevTex, 3 postscript figures, Submitted to Phys. Rev. Lett. Apr. 23, 1997 July 7, 1997: minor corrections, final accepted versio

The BAF chromatin remodelling complex is an epigenetic regulator of lineage specification in the early mouse embryo.

Dynamic control of gene expression is essential for the development of a totipotent zygote into an embryo with defined cell lineages. The accessibility of genes responsible for cell specification to transcriptional machinery is dependent on chromatin remodelling complexes such as the SWI\SNF (BAF) complex. However, the role of the BAF complex in early mouse development has remained unclear. Here, we demonstrate that BAF155, a major BAF complex subunit, regulates the assembly of the BAF complex in vivo and regulates lineage specification of the mouse blastocyst. We find that associations of BAF155 with other BAF complex subunits become enriched in extra-embryonic lineages just prior to implantation. This enrichment is attributed to decreased mobility of BAF155 in extra-embryonic compared with embryonic lineages. Downregulation of BAF155 leads to increased expression of the pluripotency marker Nanog and its ectopic expression in extra-embryonic lineages, whereas upregulation of BAF155 leads to the upregulation of differentiation markers. Finally, we show that the arginine methyltransferase CARM1 methylates BAF155, which differentially influences assembly of the BAF complex between the lineages and the expression of pluripotency markers. Together, our results indicate a novel role of BAF-dependent chromatin remodelling in mouse development via regulation of lineage specification.We are grateful to: W. Xu for an antibody against methylated BAF155; R. Kemler for an antibody against Troma1, A. Surani for TS cells, P. Almeida Coelho, S. Vuoristo and A. Hupalowska for technical assistance; M. Bredford for the CARM1 -/- line; D. Glover, M-E Torres-Padilla, P. P. Amaral, K. Miyamoto, A. Bruce, I. Bedzhov and J. Gurdon for helpful suggestions. This work was supported by The Wellcome Trust Senior Fellowship to MZG; M.P. is supported by the Darwin Trust of Edinburgh.This is the final version of the article. It first appeared from The Company of Biologists via https://doi.org/10.1242/dev.13196

Molecular Analysis of an ATP-Dependent Anion Pump

The plasmid-borne arsenical resistance operon encodes an ATP-driven oxyanion pump for the extrusion of the oxyanions arsenite, antimonite and arsenate from bacterial cells. The catalytic component of the pump, the 63 kDa ArsA protein, hydrolysesATP in the presence of its anionic substrate antimonite (SbO;). The ATP analogue 5' -p-fluorosulphonylbenzoyladenosine was used to modify the ATP binding site (s) of the ArsA protein. From sequence analysis there are two potential nucleotide binding sites. Mutations were introduced into the N-terminal site. Purified mutant proteins were catalytically inactive and incapable of binding nucleotides. Conformational changes produced upon binding of substrates to the ArsA protein were investigated by measuring the effects of substrates on trypsin inactivation. The hydrophobic 45.5 kDa ArsB protein forms the membrane anchor for the ArsA protein. The presence of the ArsA protein on purified inner membrane can be detected immunologically. In the absence of the arsB gene no ArsA is found on the membrane. Synthesis of the ArsB protein is limiting for formation of the pump. Analysis ofmRNA structure suggests a potential translational block to synthesis of the ArsB protein. Northern analysis of the ars message demonstrates rapid degradation of the mRNA in the arsB region

Epidemiological profile and clinico-pathological features of pediatric gynecological cancers at Moi Teaching & Referral Hospital, Kenya

Background: The main pediatric (0–18 years) gynecologic cancers include stromal carcinomas (juvenile granulosa cell tumors and Sertoli-Leydig cell tumors), genital rhabdomyosarcomas and ovarian germ cell. Outcomes depend on time of diagnosis, stage, tumor type and treatment which can have long-term effects on the reproductive career of these patients. This study seeks to analyze the trends in clinical-pathologic presentation, treatment and outcomes in the cases seen at our facility. This is the first paper identifying these cancers published from sub-Saharan Africa. Method: Retrospective review of clinico-pathologic profiles and treatment outcomes of pediatric gynecologic oncology patients managed at MTRH between 2010 and 2020. Data was abstracted from gynecologic oncology database and medical charts. Results: Records of 40 patients were analyzed. Most, (92.5%, 37/40) of the patients were between 10 and 18 years. Ovarian germ cell tumors were the leading histological diagnosis in 72.5% (29/40) of the patients; with dysgerminomas being the commonest subtype seen in 12 of the 37 patients (32.4%). The patients received platinum-based chemotherapy in 70% of cases (28/40). There were 14 deaths among the 40 patients (35%) Conclusion: Surgery remains the main stay of treatment and fertility-sparing surgery with or without adjuvant platinum-based chemotherapy are the standard of care with excellent prognosis following early detection and treatment initiation. LMICs face several challenges in access to quality care and that affects survival of these patients. Due to its commonality, ovarian germ cell cancers warrant a high index of suspicion amongst primary care providers attending to adnexal masses in this age group

A plasmid-encoded anion-translocating ATPase

An anion-translocating ATPase has been identified as the product of the arsenical resistance operon of resistance plasmid R773. When expressed in Escherichia coli this ATP-driven oxyanion pump catalyzes extrusion of the oxyanions arsenite, antimonite and arsenate. Maintenance of a low intracellular concentration of oxyanion produces resistance to the toxic agents. The pump is composed of two polypeptides, the products of the arsA and arsB genes. This two-subunit enzyme produces resistance to arsenite and antimonite. A third gene, arsC, expands the substrate specificity to allow for arsenate pumping and resistance

Private Sector Union Density and the Wage Premium: Past, Present, and Future

The rise and decline of private sector unionization were among the more important features of the U.S. labor market during the twentieth century. Following a dramatic spurt in unionization after passage of the depression-era National Labor Relations Act (NLRA) of 1935, union density peaked in the mid-1950s, and then began a continuous decline. At the end of the century, the percentage of private wage and salary workers who were union members was less than 10 percent, not greatly different from union density prior to the NLRA

Perineal discomfort in prostatic adenocarcinoma

We highlight the risk of missing a high-grade\ud (Gleason Grade 7/8) transition zone adenocarcinoma\ud in a patient presenting with perineal discomfort\ud on sitting