Background The reovirus λ2 protein catalyzes mRNA capping, that is, addition of a guanosine to the 5' end of each transcript in a 5'-to-5' orientation, as well as transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the N7 atom of the added guanosyl moiety and subsequently to the ribose 2'-O atom of the first template-encoded nucleotide. The structure of the human reovirus core has been solved at 3.6 Å resolution, revealing a series of domains that include a putative guanylyltransferase domain and two putative methyltransferase (MTase) domains. It has been suggested that the order of domains in the λ2 protein corresponds to the order of reactions in the pathway and that the m7G (cap 0) and the 2'-O-ribose (cap 1) MTase activities may be exerted by the MTase 1 and the MTase 2 domains, respectively. Results We show that the reovirus MTase 1 domain shares a putative active site with the structurally characterized 2'-O-ribose MTases, including vaccinia virus cap 1 MTase, whereas the MTase 2 domain is structurally similar to glycine N-MTase. Conclusions On the basis of our analysis of the structural details we propose that the previously suggested functional assignments of the MTase 1 and MTase 2 domains should be swapped.

Background PCR amplification of target molecules involves sequence specific primers that flank the region to be amplified. While this technique is generally routine, its applicability may not be sufficient to generate a desired target molecule from two separate regions involving intron /exon boundaries. For these situations, the generation of full-length complementary DNAs from two partial genomic clones becomes necessary for the family of low abundance genes. Results The first approach we used for the isolation of full-length cDNA from two known genomic clones of Hox genes was based on fusion PCR. Here we describe a simple and efficient method of amplification for homeobox D13 (HOXD13) full length cDNA from two partial genomic clones. Specific 5' and 3' untranslated region (UTR) primer pairs and website program (primer3_www.cgv0.2) were key steps involved in this process. Conclusions We have devised a simple, rapid and easy method for generating cDNA clone from genomic sequences. The full length HOXD13 clone (1.1 kb) generated with this technique was confirmed by sequence analysis. This simple approach can be utilized to generate full-length cDNA clones from available partial genomic sequences.

We evaluated the Templated Oligo with Sequencing Readout (TempO-Seq) assay for High-throughput transcriptomic screening of a small set of environmental chemicals. This assay yields sequencing reads of exactly 50 base pairs that can be rapidly aligned to generate gene counts, and is compatible with cell lysates prepared in multiwell format. The version of the TempO-Seq assay we evaluated provides nearly whole transcriptome coverage (>20,000 genes). This study encompasses 2 replicates of a 384-well plate design. The majority of wells contain U-2 OS cells exposed to 11 test chemicals at 7 different concentrations (two replicate per test chemical, concentration, and plate), and additional reference samples and controls. Controls include DMSO vehicle treatments (22 per plate). Reference samples include bulk lysate MCF7 samples (2 DMSO treated and 2 TSA treated samples per plate), reference chemical treatments (3 chemicals at single conc each, per plate), and vendor-provided reference RNA mixtures (UHRR and HBRR). This dataset is associated with the following publication: Nyffeler, J., C. Willis, D. Harris, L. Taylor, R. Judson, L. Everett, and J. Harrill. Combining phenotypic profiles and targeted RNA-Seq reveals linkages between transcriptional perturbations and chemical effects on cell morphology: retinoic acid as an example.. TOXICOLOGY AND APPLIED PHARMACOLOGY. Academic Press Incorporated, Orlando, FL, USA, 444: 116032, (2022).

An analysis of numerous Drosophila microarray experiments reveals that the genome has many large groups of adjacent genes that are expressed similarly but are not functionally related.

Background Previous kinetic investigations of fungal-peroxidase catalyzed oxidation of N-aryl hydroxamic acids (AHAs) and N-aryl-N-hydroxy urethanes (AHUs) revealed that the rate of reaction was independent of the formal redox potential of substrates. Moreover, the oxidation rate was 3–5 orders of magnitude less than for oxidation of physiological phenol substrates, though the redox potential was similar. Results To explain the unexpectedly low reactivity of AHAs and AHUs we made ab initio calculations of the molecular structure of the substrates following in silico docking in the active center of the enzyme. Conclusions AHAs and AHUs were docked at the distal side of heme in the sites formed by hydrophobic amino acid residues that retarded a proton transfer and finally the oxidation rate. The analogous phenol substrates were docked at different sites permitting fast proton transfer in the relay of distal His and water that helped fast substrate oxidation.

The attached extensive computational chemistry dataset involves detailed electronic structure (density functional theory - DFT) and kinetic calculation (master equation formalism) outputs for the reactions of isoprene and first generation oxidants with the hydroxyl radical. There are a total of 9 tabs in the Excel spreadsheet. The first two tabs provide the potential energy surfaces (PESs) of the isoprene+OH and isopOOH+OH (1st generation oxidant) reactions. The PESs are zero-point energy corrected and obtained at the M062x/maug-cc-pVTZ level of DFT. The third tab provides the reaction barriers for first and second generation 1,5-hydrogen atom shifts for two different isoprene peroxy radical isomers with several different DFT methods. The fourth and fifth tabs provide microcanonical rate constants for the reactions of isoprene and isopOOH with OH respectively. The remaining tabs give the rate constants for the 1,5-H shifts for four different isoprene peroxy radicals. The rate constants are computed using the M062x density functional and an average of 4 different DFT methods given in tab 3 for comparison. The average values are reported as the final rate constants determined by computational methods. This dataset is associated with the following publication: Piletic, I., R. Howell, L. Bartolotti, T. Kleindienst, S. Kaushik, and E. Edney. Multigenerational Theoretical Study of Isoprene Peroxy Radical 1–5-Hydrogen Shift Reactions that Regenerate HOx Radicals and Produce Highly Oxidized Molecules. JOURNAL OF PHYSICAL CHEMISTRY A. American Chemical Society, Washington, DC, USA, 123(4): 906-919, (2019).

The attached extensive computational chemistry dataset involves detailed electronic structure (density functional theory - DFT) and kinetic calculation (master equation formalism) outputs for the reactions of isoprene and first generation oxidants with the hydroxyl radical. There are a total of 9 tabs in the Excel spreadsheet. The first two tabs provide the potential energy surfaces (PESs) of the isoprene+OH and isopOOH+OH (1st generation oxidant) reactions. The PESs are zero-point energy corrected and obtained at the M062x/maug-cc-pVTZ level of DFT. The third tab provides the reaction barriers for first and second generation 1,5-hydrogen atom shifts for two different isoprene peroxy radical isomers with several different DFT methods. The fourth and fifth tabs provide microcanonical rate constants for the reactions of isoprene and isopOOH with OH respectively. The remaining tabs give the rate constants for the 1,5-H shifts for four different isoprene peroxy radicals. The rate constants are computed using the M062x density functional and an average of 4 different DFT methods given in tab 3 for comparison. The average values are reported as the final rate constants determined by computational methods. This dataset is associated with the following publication: Piletic, I., R. Howell, L. Bartolotti, T. Kleindienst, S. Kaushik, and E. Edney. Multigenerational Theoretical Study of Isoprene Peroxy Radical 1–5-Hydrogen Shift Reactions that Regenerate HOx Radicals and Produce Highly Oxidized Molecules. JOURNAL OF PHYSICAL CHEMISTRY A. American Chemical Society, Washington, DC, USA, 123(4): 906-919, (2019).

These spreadsheets contain the underlying data, calculations, and R code for the figures of the manuscript "Persistent gene expression and DNA methylation alterations linked to carcinogenic effects of dichloroacetic acid". The first tab of every spreadsheet contains descriptions of the data (metadata). For clarifying questions regarding the data, please contact Dr. Brian Chorley (EPA-ORD) at chorley.brian@epa.gov. If you require a review token for the sequencing datasets located in the GEO links (RNAseq and RRBS), please contact Dr. Chorley. This dataset is associated with the following publication: Carswell, G., J. Chamberlin, B. Bennett, P. Bushel, and B. Chorley. Persistent gene expression and DNA methylation alterations linked to carcinogenic effects of dichloroacetic acid. Frontiers in Oncology. Frontiers, Lausanne, SWITZERLAND, 14: 1389634, (2024).

Background: Genome comparisons have revealed major lateral gene transfer between the three primary kingdoms of life - Bacteria, Archaea, and Eukarya. Another important evolutionary phenomenon involves the evolutionary mobility of protein domains that form versatile multidomain architectures. We were interested in investigating the possibility of a combination of these phenomena, with an invading gene merging with a pre-existing gene in the recipient genome. Results: Complete genomes of fifteen bacteria, four archaea and one eukaryote were searched for interkingdom gene fusions (IKFs); that is, genes coding for proteins that apparently consist of domains originating from different primary kingdoms. Phylogenetic analysis supported 37 cases of IKF, each of which includes a 'native' domain and a horizontally acquired 'alien' domain. IKFs could have evolved via lateral transfer of a gene coding for the alien domain (or a larger protein containing this domain) followed by recombination with a native gene. For several IKFs, this scenario is supported by the presence of a gene coding for a second, stand-alone version of the alien domain in the recipient genome. Among the genomes investigated, the greatest number of IKFs has been detected in Mycobacterium tuberculosis, where they are almost always accompanied by a stand-alone alien domain. For most of the IKF cases detected in other genomes, the stand-alone counterpart is missing. Conclusions: The results of comparative genome analysis show that IKF formation is a real, but relatively rare, evolutionary phenomenon. We hypothesize that IKFs are formed primarily via the proposed two-stage mechanism, but other than in the Actinomycetes, in which IKF generation seems to be an active, ongoing process, most of the stand-alone intermediates have been eliminated, perhaps because of functional redundancy.

Results from Quantitative Interspecies Thermal Shift Assay (QITSA) and molecular docking. This dataset is associated with the following publication: Han, J., J. Fu, J. Sun, D.R. Hall, D. Yang, D. Blatz, K. Houck, C. Ng, J. Doering, C. LaLone, and H. Peng. Quantitative Chemical Proteomics Reveals Interspecies Variations on Binding Schemes of L-FABP with Perfluorooctanesulfonate. Nature Communications. Nature Publishing Group, London, UK, 55(13): 9012-9023, (2021).