PRMT7 Is a Member of the Protein Arginine Methyltransferase Family with a Distinct Substrate Specificity

We have identified a mammalian arginine N-methyltransferase, PRMT7, that can catalyze the formation of ω-N^G-monomethylarginine in peptides. This protein is encoded by a gene on human chromosome 16q22.1 (human locus AK001502). We expressed a full-length human cDNA construct in Escherichia coli as a glutathione S-transferase (GST) fusion protein. We found that GST-tagged PRMT7 catalyzes the S-adenosyl-[methyl-^3H]-L-methionine-dependent methylation of the synthetic peptide GGPGGRGGPGG-NH_2 (R1). The radiolabeled peptide was purified by high-pressure liquid chromatography and acid hydrolyzed to free amino acids. When the hydrolyzed products were separated by high-resolution cation-exchange chromatography, we were able to detect one tritiated species which co-migrated with an ω-N^G-monomethylarginine standard. Surprisingly, GST-PRMT7 was not able to catalyze the in vitro methylation of a GST-fibrillarin (amino acids 1–148) fusion protein (GST-GAR), a methyl-accepting substrate for the previously characterized PRMT1, PRMT3, PRMT4, PRMT5, and PRMT6 enzymes. Nor was it able to methylate myelin basic protein or histone H2A, in vitro substrates of PRMT5. This specificity distinguishes PRMT7 from all of the other known arginine methyltransferases. An additional unique feature of PRMT7 is that it seems to have arisen from a gene duplication event and contains two putative AdoMet-binding motifs. To see if both motifs were necessary for activity, each putative domain was expressed as a GST-fusion and tested for activity with peptides R1 and R2 (acetyl-GGRGG-NH_2). These truncated proteins were enzymatically inactive, suggesting that both domains are required for functionality

High yield non-detergent isolation of photosystem I-light-harvesting chlorophyll II membranes from spinach thylakoids: Implications for the organization of the PS I antennae in higher plants

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Styrene-maleic acid copolymer was used to effect a non-detergent partial solubilization of thylakoids from spinach. A high density membrane fraction, which was not solubilized by the copolymer, was isolated and was highly enriched in the Photosystem (PS) I-light-harvesting chlorophyll (LHC) II supercomplex and depleted of PS II, the cytochrome b6/f complex, and ATP synthase. The LHC II associated with the supercomplex appeared to be energetically coupled to PS I based on 77 K fluorescence, P700 photooxidation, and PS I electron transport light saturation experiments. The chlorophyll (Chl) a/b ratio of the PS I-LHC II membranes was 3.2 ± 0.9, indicating that on average, three LHC II trimers may associate with each PS I. The implication of these findings within the context of higher plant PS I antenna organization is discussed

Photoheterotrophic growth of Physcomitrella patens

© Springer-Verlag Berlin Heidelberg 2013. Physcomitrella patens is a model bryophyte representing an early land plant in the green plant lineage. This organism possesses many advantages as a model organism. Its genome has been sequenced, its predominant life cycle stage is the haploid gametophyte, it is readily transformable and it can integrate transformed DNA into its genome by homologous recombination. One limitation for the use of P. patens in photosynthesis research is its reported inability to grow photoheterotrophically, in the presence of sucrose and the Photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which prevents linear photosynthetic electron transport. In this communication we describe the facile isolation of a P. patens strain which can grow photoheterotrophically. Additionally, we have examined a number of photosynthetic parameters for this strain grown under photoautotrophic, mixotrophic (in the presence of sucrose) and photoheterotrophic conditions, as well as the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-inhibited state. The ability to grow P. patens photoheterotrophically should significantly facilitate its use in photosynthetic studies

Context and conformation dictate function of a transcription antitermination switch

In bacteriophage λ, transcription elongation is regulated by the N protein, which binds a nascent mRNA hairpin (termed boxB) and enables RNA polymerase to read through distal terminators. We have examined the structure, energetics and in vivo function of a number of N−boxB complexes derived from in vitro protein selection. Trp18 fully stacks on the RNA loop in the wild-type structure, and can become partially or completely unstacked when the sequence context is changed three or four residues away, resulting in a recognition interface in which the best binding residues depend on the sequence context. Notably, in vivo antitermination activity correlates with the presence of a stacked aromatic residue at position 18, but not with N−boxB binding affinity. Our work demonstrates that RNA polymerase responds to subtle conformational changes in cis-acting regulatory complexes and that approximation of components is not sufficient to generate a fully functional transcription switch

Complete Genome Sequence and Comparative Metabolic Profiling of the Prototypical Enteroaggregative Escherichia coli Strain 042

Background \ud Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation. \ud \ud Methods \ud In this study we determined the complete genomic sequence of E. coli 042, the prototypical member of the enteroaggregative E. coli, which has been shown to cause disease in volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing previously uncharacterised virulence factors including a variety of secreted proteins and a capsular polysaccharide biosynthetic locus. In addition, by using Biolog™ Phenotype Microarrays we have provided a full metabolic profiling of E. coli 042 and the non-pathogenic lab strain E. coli K-12. We have highlighted the genetic basis for many of the metabolic differences between E. coli 042 and E. coli K-12. \ud \ud Conclusion \ud This study provides a genetic context for the vast amount of experimental and epidemiological data published thus far and provides a template for future diagnostic and intervention strategies

Characterization of impact pile driving signals during installation of offshore wind turbine foundations

Author Posting. © Acoustical Society of America, 2020. This article is posted here by permission of Acoustical Society of America for personal use, not for redistribution. The definitive version was published in Journal of the Acoustical Society of America 147(4), (2020): 2323, doi:10.1121/10.0001035.Impact pile driving creates intense, impulsive sound that radiates into the surrounding environment. Piles driven vertically into the seabed generate an azimuthally symmetric underwater sound field whereas piles driven on an angle will generate an azimuthally dependent sound field. Measurements were made during pile driving of raked piles to secure jacket foundation structures to the seabed in waters off the northeastern coast of the U.S. at ranges between 500 m and 15 km. These measurements were analyzed to investigate variations in rise time, decay time, pulse duration, kurtosis, and sound received levels as a function of range and azimuth. Variations in the radiated sound field along opposing azimuths resulted in differences in measured sound exposure levels of up to 10 dB and greater due to the pile rake as the sound propagated in range. The raked pile configuration was modeled using an equivalent axisymmetric FEM model to describe the azimuthally dependent measured sound fields. Comparable sound level differences in the model results confirmed that the azimuthal discrepancy observed in the measured data was due to the inclination of the pile being driven relative to the receiver.This paper was presented at the fifth International Meeting on The Effects of Noise on Aquatic Life held in Den Haag, July 2019. Study concept, oversight, and funding for the experiment were provided by the U.S. Department of the Interior, Bureau of Ocean Energy Management (BOEM), Environmental Studies Program, Washington, DC, under Contract No. M15PC00002, Task Order M16PD00025. Collaborators in this project include Randy Gallien and Anwar Khan (HDR, Inc.).2020-10-1