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Thomas C Evans

Publications and source records attributed to Thomas C Evans.

14 recordsLinked to original sources
PubMed2006

The Sm proteins regulate germ cell specification during early C. elegans embryogenesis.

Sm and Sm-like proteins are core components of the splicesome but have other functions distinct from pre-mRNA processing. Here, we show that Sm proteins also regulate germ cell specification during early C. elegans embryogenesis. SmE and SmG were required to maintain transcriptional quiescence in embryonic germ cell precursors. In addition, depletion of SmE inhibited expression of the germ lineage-specific proteins PIE-1, GLD-1, and NOS-2, but did not affect maintenance of several maternal mRNAs. PIE-1 had previously been shown to activate transcriptional silencing and NOS-2 expression. We found that PIE-1 also promotes GLD-1 expression by a process that is independent of transcriptional silencing. Thus, Sm proteins could control transcriptional silencing and maternal protein expression by regulating PIE-1. However, loss of SmE function also caused defects in P granule localization and premature division in early germline blastomeres, processes that are independent of PIE-1 function. Therefore, the Sm proteins control multiple aspects of germ cell precursor development. Because depletion of several other core splicing factors did not affect these events, these Sm functions are likely distinct from pre-mRNA splicing. Sm family proteins assemble into ribonucleoprotein complexes (RNPs) that control RNA activities. We suggest that novel Sm RNPs directly or indirectly influence posttranscriptional control of maternal mRNAs to promote germ cell specification in the early C. elegans embryo.

Animals↗
PubMed2005

Sequence specificity and role of proximal amino acids of the histone H3 tail on catalysis of murine G9A lysine 9 histone H3 methyltransferase.

The activity of recombinant murine G9a toward lysine 9 of histone H3 was investigated. GST fusion proteins containing various lengths of the histone H3 amino-terminal tail were used as substrates in the presence of recombinant G9a enzyme and AdoMet cosubstrate. The minimal substrate methylated by G9a contained seven amino acids (TARKSTG) of the histone H3 tail. Furthermore, mutational analysis of the minimal substrate was performed to identify the amino acids essential for G9a-mediated methylation. All amino acids except Thr-11 were indispensable for the methylation reaction. Steady-state kinetic analysis of the wild-type and histone H3 point mutants, lysine 4 changed to alanine (K4A) or lysine 27 changed to alanine (K27A), with purified G9a revealed similar catalytic efficiency but a reduction in turnover number (k(cat)) from 78 to 58 h(-)(1). G9a methylated synthetic peptide substrates containing the first 13 amino acids of histone H3 efficiently, although methylation, acetylation, or mutation of proximal Lys-4 amino acids reduced Lys-9 methylation. The k(cat) for wild-type peptide substrate vs Lys-4 acetyl- or trimethyl-modified peptide were 88 and 32 h(-)(1), respectively, and the K(m) for the peptides varied from 0.6 to 2.2 muM, resulting in a large difference (15-91) in catalytic efficiency. Ser-10 or Thr-11 phosphorylation resulted in poor methylation by G9a. Immunoprecipitation of unmodified and Ser-10 and Thr-11 phosphorylated histone H3 displayed mostly Lys-4 dimethylation. Dimethylated Lys-9 was reduced in Ser-10 and Thr-11 immunoprecipitated phosphorylated histones as compared to nonphosphorylated H3. In an immunocytochemical assay, GFP fusion SUV39H1 or G9a did not colocalize with phosphorylated histone H3. Thus, Ser-10/Thr-11 phosphorylation impairs Lys-9 methylation. These data suggest that the sequence context of the modified residue affects G9a activity and the modification in the proximal amino acids influences methylation.

Amino Acid Sequence↗
PubMed2005

Crystal structures of an intein from the split dnaE gene of Synechocystis sp. PCC6803 reveal the catalytic model without the penultimate histidine and the mechanism of zinc ion inhibition of protein splicing.

The first naturally occurring split intein was found in the dnaE gene of Synechocystis sp. PCC6803 and belongs to a subclass of inteins without a penultimate histidine residue. We describe two high-resolution crystal structures, one derived from an excised Ssp DnaE intein and the second from a splicing-deficient precursor protein. The X-ray structures indicate that His147 in the conserved block F activates the side-chain N(delta) atom of the intein C-terminal Asn159, leading to a nucleophilic attack on the peptide bond carbonyl carbon atom at the C-terminal splice site. In this process, Arg73 appears to stabilize the transition state by interacting with the carbonyl oxygen atom of the scissile bond. Arg73 also seems to substitute for the conserved penultimate histidine residue in the formation of an oxyanion hole, as previously identified in other inteins. The finding that the precursor structure contains a zinc ion chelating the highly conserved Cys160 and Asp140 reveals the structural basis of Zn2+-mediated inhibition of protein splicing. Furthermore, it is of interest to observe that the carbonyl carbon atom of Asn159 and N(eta) of Arg73 are 2.6 angstroms apart in the free intein structure and 10.6 angstroms apart in the precursor structure. The orientation change of the aromatic ring of Tyr-1 following the initial acyl shift may be a key switching event contributing to the alignment of Arg73 and the C-terminal scissile bond, and may explain the sequential reaction property of the Ssp DnaE intein.

Bacterial Proteins↗
PubMed2005

Recent advances in protein splicing: manipulating proteins in vitro and in vivo.

Protein splicing is an intricate self-catalyzed protein rearrangement that converts an inactive protein precursor to biologically active proteins. In the past decade, mechanistic studies and extensive engineering of the naturally occurring protein splicing elements, termed inteins, has led to the development of numerous novel technologies. These intein-based methodologies permit in vitro and in vivo protein processing in ways previously not possible using traditional biochemical and genetic approaches. Inteins have been utilized in the production of protein and peptide arrays, as molecular switches and in the reconstitution of functional proteins by split-gene techniques.

Animals↗
PubMed2005

Protein splicing elements and plants: from transgene containment to protein purification.

Protein splicing elements, termed inteins, have been discovered in all the domains of life. Basic research on inteins has led to a greater understanding of how they mediate the protein splicing process. Because inteins are natural protein engineering elements they have been harnessed for use in a number of applications, including protein purification, protein semisynthesis, and in vivo and in vitro protein modifications. This review focuses on the use of inteins in plants. A split-gene technique utilizes inteins to reconstitute the activity of a transgene product with the goal of limiting the spread of transgenes from a genetically modified plant to a weedy relative. Furthermore, merging the intein tag for protein purification with the large protein yields possible with plants has the potential to produce pharmaceutically important proteins. Finally, relevant techniques that may be used in plants in the future are discussed.

Acetolactate Synthase↗
PubMed2004

Mutational analysis of protein splicing, cleavage, and self-association reactions mediated by the naturally split Ssp DnaE intein.

The ability to separately purify the naturally split Synechocystis sp. PCC6803 (Ssp) DnaE intein domains has allowed detailed examination of both universal and Ssp DnaE intein-specific steps in the protein splicing pathway. By engineering substitutions at both the +1 and penultimate intein positions, we have further characterized intein reaction kinetics in this system. Replacement of the crucial +1Cys with serine decreased N-terminal cleavage and trans-splicing rates; however, this substitution did not prevent splicing or the ability of ZnCl2 to inhibit it. Substitution of the penultimate intein residue (alanine) with a typically conserved histidine did not increase the rate or extent of trans-splicing or cleavage under typical assay conditions. Despite the observation that this histidine aids in asparagine cyclization for other inteins, it did not encourage C-terminal cleavage for the Ssp DnaE intein or uncouple it from N-terminal cleavage. Both the +1Ser and Ala to His mutants were insensitive to ZnCl2 during trans-cleavage experiments, uncoupling a previously linked inhibition in asparagine cyclization from an inhibition in trans-thioesterification detected for the wild-type intein.

Alanine↗
PubMed2004

An improved method for utilization of peptide substrates for antibody characterization and enzymatic assays.

Synthetic peptides have become an important tool in antibody production and enzyme characterization. The small size of peptides, however, has hindered their use in assays systems, such as Western blots, and as immunogens. Here, we present a facile method to improve the properties of peptides for multiple applications by ligating the peptides to intein-generated carrier proteins. The stoichiometric ligation of peptide and carrier achieved by intein-mediated protein ligation (IPL) results in the ligation product migrating as a single band on a SDS-PAGE gel. The carrier proteins, HhaI methylase (M.HhaI) and maltose-binding protein (MBP), were ligated to various peptides; the ligated carrier-peptide products gave sharp, reproducible bands when used as positive controls for antibodies raised against the same peptides during Western blot analysis. We further show that ligation of the peptide antigens to a different thioester-tagged carrier protein, paramyosin, produced immunogens for the production of antisera in rabbits or mice. Furthermore, we demonstrate the generation of a substrate for enzymatic assays by ligating a peptide containing the phosphorylation site for Abl protein tyrosine kinase to a carrier protein. This carrier-peptide protein was used as a kinase substrate that could easily be tested for phosphorylation using a phosphotyrosine antibody in Western blot analysis. These techniques do not require sophisticated equipment, reagents, or skills thereby providing a simple method for research and development.

Animals↗
PubMed2003

Zinc ion effects on individual Ssp DnaE intein splicing steps: regulating pathway progression.

Use of the naturally split, self-splicing Synechocystis sp. PCC6803 DnaE intein permits separate purification of the N- and C-terminal intein domains. Otherwise spontaneous intein-mediated reactions can therefore be controlled in vitro, allowing detailed study of intein kinetics. Incubation of the Ssp DnaE intein with ZnCl(2) inhibited trans splicing, hydrolysis-mediated N-terminal trans cleavage, and C-terminal trans cleavage reactions. Maximum inhibition of the splicing reaction was achieved at equal molar concentrations of ZnCl(2) and intein domains, suggesting a 1:1 metal ion:intein binding stoichiometry. Mutation of the (+)1 cysteine residue to valine (C(+)1V) alleviated the inhibitory effects of ZnCl(2). Valine substitution in the absence of ZnCl(2) blocked trans splicing and decreased C-terminal cleavage kinetics in a manner similar to that of the native (+)1 cysteine in the presence of ZnCl(2). These data are consistent with Zn(2+)-mediated inhibition of the Ssp DnaE intein via chelation of the (+)1 cysteine residue. N-Terminal trans cleavage can occur via both spontaneous hydrolysis and nucleophilic (e.g., DTT) attack. Comparative examination of N-terminal cleavage rates using amino acid substitution (C(+)1V) and Zn(2+)-mediated inhibition permitted the maximum contribution of hydrolysis to overall N-terminal cleavage kinetics to be determined. Stable intermediates consisting of the associated intein domains were detected by PAGE and provided evidence of a rapid C-terminal cleavage step. Acute control of the C-terminal reaction was achieved by the rapid reversal of Zn(2+)-mediated inhibition by EDTA. By inhibiting both the splicing pathway and spontaneous hydrolysis with Zn(2+), reactants can be diverted from the trans splicing to the trans cleavage pathway where DTT and EDTA can regulate N- and C-terminal cleavage, respectively.

Amino Acid Sequence↗
PubMed2003

Protein trans-splicing in transgenic plant chloroplast: reconstruction of herbicide resistance from split genes.

Inteins are intervening protein sequences that undergo self-excision from a precursor protein with concomitant joining of the flanking sequences. Here, we demonstrate intein trans-splicing in Nicotiana tabacum chloroplasts by using the naturally split Ssp DnaE intein. Trans-splicing occurred whether both intein fragments were encoded in the chloroplast or were separated into the chloroplast and nuclear genomes. A biolistic approach was used to integrate two fusion genes, one encoding aminoglycoside-3-adenyltransferase (aadA) and the first 123 aa of the Ssp DnaE intein (In) and the other encoding 36 C-terminal amino acid residues of the Ssp DnaE intein (Ic) and soluble modified green fluorescent protein (smGFP) into N. tabacum plastids. Expression of these gene fragments in the chloroplast resulted in ligated aadA-smGFP due to In-Ic-mediated trans-splicing. Furthermore, an N-terminal portion of the herbicide resistance gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) containing a chloroplast localization signal fused to In (EPSPSn-In) was integrated into the nuclear DNA of N. tabacum by using Agrobacterium tumefaciens-mediated transformation. The remaining EPSPS gene fragment (EPSPSc) fused to Ic (Ic-EPSPSc) was integrated into the chloroplast genome by homologous recombination. Western blot analysis of cell extracts from these plants showed a full-length EPSPS, demonstrating that the EPSPSn-In gene product migrated to the chloroplast and underwent trans-splicing. Furthermore, these transgenic plants displayed improved resistance to the herbicide N-(phosphonomethyl)glycine (glyphosate) when compared with wild-type N. tabacum.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2003

Translational repression of a C. elegans Notch mRNA by the STAR/KH domain protein GLD-1.

In C. elegans, the Notch receptor GLP-1 is localized within the germline and early embryo by translational control of glp-1 mRNA. RNA elements in the glp-1 3'untranslated region (3' UTR) are necessary for repression of glp-1 translation in germ cells, and for localization of translation to anterior cells of the early embryo. The direct regulators of glp-1 mRNA are not known. Here, we show that a 34 nucleotide region of the glp-1 3' UTR contains two regulatory elements, an element that represses translation in germ cells and posterior cells of the early embryo, and an element that inhibits repressor activity to promote translation in the embryo. Furthermore, we show that the STAR/KH domain protein GLD-1 binds directly and specifically to the repressor element. Depletion of GLD-1 activity by RNA interference causes loss of endogenous glp-1 mRNA repression in early meiotic germ cells, and in posterior cells of the early embryo. Therefore, GLD-1 is a direct repressor of glp-1 translation at two developmental stages. These results suggest a new function for GLD-1 in regulating early embryonic asymmetry. Furthermore, these observations indicate that precise control of GLD-1 activity by other regulatory factors is important to localize this Notch receptor, and contributes to the spatial organization of Notch signaling.

Animals↗
PubMed2002

A novel function for the Sm proteins in germ granule localization during C. elegans embryogenesis.

General mRNA processing factors are traditionally thought to function only in the control of global gene expression. Here we show that the Sm proteins, core components of the splicesome, also regulate germ granules during early C. elegans development. Germ granules are large cytoplasmic particles that localize to germ cells and their precursors during embryogenesis of diverse organisms. In C. elegans, germ granules, called P granules, are segregated to the germline precursor cells during embryogenesis by asymmetric cell division, and they remain in germ cells at all stages of development. We found that at least some Sm proteins are components of P granules. Moreover, disruption of Sm activity caused defects in P granule localization to the germ cell precursors during early embryogenesis. In contrast, loss of other splicing factor activities had no effect on germ granule control in the embryo. These observations suggest that the Sm proteins control germ granule integrity and localization in the early C. elegans embryo and that this role is independent of pre-mRNA splicing. Thus, a highly conserved splicing factor may have been adapted to control both snRNP biogenesis and the localization of components important for germ cell function.

Animals↗
PubMedDate not supplied

National Quality Forum 30 safe practices: priority and progress in Iowa hospitals.

The National Quality Forum (NQF) recently released a list of "30 Safe Practices" that were identified as relevant for all hospitals. The purpose of the present analysis was to assess hospitals' perceptions of each of the NQF 30 Safe Practices in terms of priority and progress. One hundred of Iowa's hospitals (86%) completed a survey. The highest progress ratings were for items involving hand washing, unit-dose medication dispensing, influenza vaccinations, implementing protocols to prevent wrong-site procedures, and standardized methods for labeling and storing medications. The lowest progress ratings were for intensive care units staffed by intensivists and implementing a computerized provider order entry system. Overall, safe practices that have been recommended for some time had higher priority and progress ratings. Most safe practices were equally endorsed by large and small hospitals, suggesting that the NQF goal of identifying safe hospital practices may be attainable for most of the safe practices.

Data Collection↗