scholarly journals Expression and characterization of a human pyruvate carboxylase variant by retroviral gene transfer

2003 ◽  
Vol 370 (1) ◽  
pp. 275-282 ◽  
Author(s):  
Mary Anna CARBONE ◽  
Brian H. ROBINSON
Keyword(s):  
Steady State ◽  
Pyruvate Carboxylase ◽  
Expression System ◽  
Type A ◽  
Immunoblot Analysis ◽  
Retroviral Gene Transfer ◽  
Reverse Transcription Pcr ◽  
Steady State Levels ◽  
The Stability

Type A pyruvate carboxylase (PC) deficiency presents mainly in the Amerindian population, specifically the Ojibwa, Cree and Micmac tribes of the Algonquin-speaking peoples. The gene for PC contains a homozygous founder mutation (G1828→A) that results in an Ala610→Thr amino acid substitution in Ojibwa with Type A PC deficiency. The mutation is located in the highly conserved pyruvate-binding domain of PC. The present paper describes a retroviral expression system for human PC used to analyse the effects of this mutation. We show, through immunoblot analysis, PC enzyme activity assays, reverse-transcription PCR and mitochondrial-import experiments, that this mutation is disease-causing in the Ojibwa population owing to its decreased catalytic activity, decreased steady-state levels of expression and inefficient import into the mitochondria. Our data suggest that this mutation may affect the stability of the protein, resulting in decreased steady-state levels of expression, and that it may also affect the secondary structure of the protein during the import process, thereby inhibiting proper translocation into the mitochondria, where PC is active.

scholarly journals Characterization of the Proteasome Accessory Factor (paf) Operon in Mycobacterium tuberculosis

2007 ◽  
Vol 189 (8) ◽  
pp. 3044-3050 ◽  
Author(s):  
Richard A. Festa ◽  
Michael J. Pearce ◽  
K. Heran Darwin
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Steady State ◽  
Unknown Function ◽  
The Other ◽  
Accessory Factor ◽  
Reactive Nitrogen Intermediates ◽  
Steady State Levels ◽  
The Stability ◽  
Transposon Insertions

ABSTRACT In a previous screen for Mycobacterium tuberculosis mutants that are hypersusceptible to reactive nitrogen intermediates (RNI), two genes associated with the M. tuberculosis proteasome were identified. One of these genes, pafA (proteasome accessory factor A), encodes a protein of unknown function. In this work, we determined that pafA is in an operon with two additional genes, pafB and pafC. In order to assess the contribution of these genes to RNI resistance, we isolated mutants with transposon insertions in pafB and pafC. In contrast to the pafA mutant, the pafB and pafC mutants were not severely sensitized to RNI, but pafB and pafC were nonetheless required for full RNI resistance. We also found that PafB and PafC interact with each other and that each is likely required for the stability of the other protein in M. tuberculosis. Finally, we show that the presence of PafA, but not PafB or PafC, regulates the steady-state levels of three proteasome substrates. Taken together, these data demonstrate that PafA, but not PafB or PafC, is critical for maintaining the steady-state levels of known proteasome substrates, whereas all three proteins appear to play a role in RNI resistance.

scholarly journals Functional characterization of the human tRNA methyltransferases TRMT10A and TRMT10B

2020 ◽  
Vol 48 (11) ◽  
pp. 6157-6169 ◽  
Author(s):  
Elisa Vilardo ◽  
Fabian Amman ◽  
Ursula Toth ◽  
Annika Kotter ◽  
Mark Helm ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Steady State ◽  
Human Genome ◽  
Functional Significance ◽  
Functional Characterization ◽  
Trna Methyltransferase ◽  
Steady State Levels

Abstract The TRM10 family of methyltransferases is responsible for the N1-methylation of purines at position 9 of tRNAs in Archaea and Eukarya. The human genome encodes three TRM10-type enzymes, of which only the mitochondrial TRMT10C was previously characterized in detail, whereas the functional significance of the two presumably nuclear enzymes TRMT10A and TRMT10B remained unexplained. Here we show that TRMT10A is m1G9-specific and methylates a subset of nuclear-encoded tRNAs, whilst TRMT10B is the first m1A9-specific tRNA methyltransferase found in eukaryotes and is responsible for the modification of a single nuclear-encoded tRNA. Furthermore, we show that the lack of G9 methylation causes a decrease in the steady-state levels of the initiator tRNAiMet-CAT and an alteration in its further post-transcriptional modification. Our work finally clarifies the function of TRMT10A and TRMT10B in vivo and provides evidence that the loss of TRMT10A affects the pool of cytosolic tRNAs required for protein synthesis.

scholarly journals Development of a High-Throughput Cell-Based Reporter Assay to Identify Stabilizers of Tumor Suppressor Pdcd4

2009 ◽  
Vol 15 (1) ◽  
pp. 21-29 ◽  
Author(s):  
Johanna S. Blees ◽  
Tobias Schmid ◽  
Cheryl L. Thomas ◽  
Alyson R. Baker ◽  
Lauren Benson ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
High Throughput ◽  
Expression System ◽  
Control Cell ◽  
Cell System ◽  
Luciferase Expression ◽  
The Novel ◽  
Pure Compounds ◽  
The Stability

The novel tumor suppressor Pdcd4 affects tumorigenesis by inhibiting translation. Pdcd4 is phosphorylated and subsequently lost by proteasomal degradation in response to tumor-promoting conditions. Here, the authors describe the development of a reporter cell system to monitor the stability of Pdcd4. The phosphorylation-dependent degradation domain (“target”) or an adjacent (“off-target”) region of Pdcd4 was cloned into a luciferase expression system. The target constructs were responsive to Pdcd4 degrading conditions (e.g., TPA, p70S6K1 overactivation), whereas the off-target constructs remained stable. The system was optimized for and shown to be reliable in a high-throughput compatible 384-well format. Screening of 15,275 pure compounds resulted in a hit rate of 0.30% (>" xbd="1092" xhg="1069" ybd="1185" yhg="1147"/>50% inhibition of TPA-induced loss of signal, confirmed by reassay). Among the hits were inhibitors of previously identified critical signaling events for TPA-induced Pdcd4 degradation. One compound was identified to be nonspecific using the off-target control cell line. Screening of 135,678 natural product extracts yielded 42 confirmed, specific hits. Z′ averaged 0.58 across 446 plates. Further characterization of active natural products and synthetic compounds is expected to identify novel Pdcd4 stabilizers that may be useful in targeting translation to prevent or treat cancers. (Journal of Biomolecular Screening 2010:21-29)

scholarly journals UTP-Dependent and -Independent Pathways of mRNA Turnover in Trypanosoma brucei Mitochondria

2000 ◽  
Vol 20 (7) ◽  
pp. 2308-2316 ◽  
Author(s):  
Kevin T. Militello ◽  
Laurie K. Read
Keyword(s):  
Steady State ◽  
Trypanosoma Brucei ◽  
Mitochondrial Gene ◽  
Rna Stability ◽  
Degradation Pathway ◽  
Mitochondrial Rna ◽  
In Organello ◽  
Steady State Levels ◽  
The Stability ◽  
Mrna Polyadenylation

ABSTRACT Although primary transcripts are polycistronic in the mitochondria of Trypanosoma brucei, steady-state levels of mature, monocistronic RNAs change throughout the parasitic life cycle. This indicates that steady-state RNA abundance is controlled by posttranscriptional mechanisms involving differential RNA stability. In this study, in organello pulse-chase labeling experiments were used to analyze the stability of different T. brucei mitochondrial RNA populations. In this system, total RNA and rRNA are stable for many hours. In contrast, mRNAs can be degraded by two biochemically distinct turnover pathways. The first pathway results in the rapid degradation of mRNA (half-life [t 1/2] of 11 to 18 min) and is dependent upon the presence of an mRNA poly(A) tail. Remarkably, this pathway also requires the addition of UTP and therefore is termed UTP dependent. The second pathway results in slow turnover of mitochondrial mRNA (t 1/2 of ∼3 h) and is not dependent upon the presence of an mRNA poly(A) tail or the addition of exogenous UTP. In summary, these results demonstrate the presence of a novel, UTP-dependent degradation pathway for T. bruceimitochondrial mRNAs and reveal an unprecedented role for both UTP and mRNA polyadenylation in T. brucei mitochondrial gene expression.

scholarly journals Different Processing of an mRNA Species inBacillus subtilis and Escherichia coli

2000 ◽  
Vol 182 (3) ◽  
pp. 689-695 ◽  
Author(s):  
Martin Persson ◽  
Elisabeth Glatz ◽  
Blanka Rutberg
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Inverted Repeat ◽  
Fusion Transcript ◽  
Temperature Sensitive ◽  
Rnase Iii ◽  
Transcription Terminator ◽  
E Coli ◽  
Steady State Levels ◽  
The Stability

ABSTRACT Expression of the Bacillus subtilis glpD gene, which encodes glycerol-3-phosphate (G3P) dehydrogenase, is controlled by termination or antitermination of transcription. The untranslated leader sequence of glpD contains an inverted repeat that gives rise to a transcription terminator. In the presence of G3P, the antiterminator protein GlpP binds toglpD leader mRNA and promotes readthrough of the terminator. Certain mutations in the inverted repeat of theglpD leader result in GlpP-independent, temperature-sensitive (TS) expression of glpD. The TS phenotype is due to temperature-dependent degradation of theglpD mRNA. In the presence of GlpP, theglpD mRNA is stabilized. glpDleader-lacZ fusions were integrated into the chromosomes ofB. subtilis and Escherichia coli. Determination of steady-state levels of fusion mRNA in B. subtilis showed that the stability of the fusion mRNA is determined by theglpD leader part. Comparison of steady-state levels and half-lives of glpD leader-lacZ fusion mRNA inB. subtilis and E. coli revealed significant differences. A glpD leader-lacZ fusion transcript that was unstable in B. subtilis was considerably more stable in E. coli. GlpP, which stabilizes the transcript in B. subtilis, did not affect its stability in E. coli. Primer extension analysis showed that theglpD leader-lacZ fusion transcript is processed differently in B. subtilis and in E. coli. The dominating cleavage site in E. coli was barely detectable in B. subtilis. This site was shown to be a target ofE. coli RNase III.

The Characterization of the Stability of Gel Ink by Rheological Method

2013 ◽  
Vol 816-817 ◽  
pp. 660-664
Author(s):  
Wang Zhao Yao ◽  
Wei Chao Wang ◽  
Ai Ping Chen ◽  
Hui Shen ◽  
Hong Fei Hao ◽  
...  
Keyword(s):  
Steady State ◽  
Shear Rate ◽  
Dynamic Stress ◽  
Experimental Methods ◽  
Rheological Parameters ◽  
Time Scanning ◽  
Rheological Method ◽  
The Stability ◽  
Dynamic Time

The rheological performance of gel ink was investigated by the experimental methods of steady-state shear rate scanning, dynamic stress scanning and dynamic time scanning. The results show that some rheological parameters (such asn,K,τ0, and ect.) can be used to characterize the stability of gel ink.

scholarly journals Characterization of Baculovirus Repeated Open Reading Frames (bro) in Bombyx moriNucleopolyhedrovirus

1999 ◽  
Vol 73 (12) ◽  
pp. 10339-10345 ◽  
Author(s):  
Wonkyung Kang ◽  
Masataka Suzuki ◽  
Evgueni Zemskov ◽  
Keiju Okano ◽  
Susumu Maeda
Keyword(s):  
Immunohistochemical Analysis ◽  
Immunoblot Analysis ◽  
Open Reading Frames ◽  
Start Codon ◽  
Sequence Analyses ◽  
Reverse Transcription Pcr ◽  
Double Deletion ◽  
Viral Factor ◽  
Reading Frames

ABSTRACT The baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) contains five related open reading frames (ORFs). Recent sequence analyses of several other baculovirus genomes reveal that these ORFs belong to a unique multigene family called the baculovirus repeated ORFs (bro) family. Here we have characterized these five genes from BmNPV at the transcriptional and translational levels. Reverse transcription-PCR and primer extension analyses indicated that transcription of all bro genes occurs by 2 to 4 h postinfection (p.i.) and reaches maximal levels between at 8 and 12 h p.i. Transcription of all genes is initiated between 50 and 70 nucleotides upstream of the start codon, at a characteristic C(T)AGT motif. Expression of a cat reporter gene under the control of each bro promoter provides evidence that a viral factor(s) is required for the transcription of all brogenes. Immunoblot analysis indicated that a population of BRO proteins is produced vigorously between at 8 and 14 h p.i. Immunohistochemical analysis by confocal microscopy showed that BRO proteins are localized in both the nucleus and the cytoplasm at 8 h p.i. Four BmNPV mutants, in which the bro-a,bro-b, bro-c, and bro-e genes were individually inactivated, were successfully isolated. However, exhaustive efforts failed to isolate a bro-d-deficient mutant. Similarly, it was not possible to isolate a double-deletionbro-a bro-c mutant. The bro-d gene may play an irreplaceable functional role(s) during viral infection, whilebro-a and bro-c may functionally complement each other.

scholarly journals χ-maps and Hypsometric Analysis for River Basin Management and Prioritization: The Case of Bohol River Basins, Central Philippines

2021 ◽  
Author(s):  
Imelida Genson Torrefranca ◽  
Roland Emerito S Otadoy ◽  
Alejandro F Tongco
Keyword(s):  
Steady State ◽  
River Basin ◽  
River Basin Management ◽  
River Channel ◽  
Stream Networks ◽  
Basin Management ◽  
New Approach ◽  
Hypsometric Analysis ◽  
The Stability

Abstract Topographic indices represent and simplify complex surficial processes fundamental to characterizing landform dynamics and to prioritizing river basin conservation and management goals. A method of characterizing landform dynamics integrating the steady-state river channel elevation and the hypsometric analysis is presented. The chi (χ) metric, a proxy for the steady-state river channel elevation, gages the stability of drainage divides while hypsometric analysis quantifies the stages of basin geological development. Using a 30m SRTM DEM and the TopoToolbox tool in MATLAB, χ - values along stream networks are computed. At the channel heads of opposing stream networks of a divide section, equal χ – values indicate a stable divide while across difference in χ - values suggest unstable divide with a potential to migrate from low χ – values towards the high χ – values side of the divide. To classify the degree of potential divide mobility, the quantity called mean chi difference (χmd) is proposed. The features of the aggressive and victim river streams are visually differentiated using their elevation profiles, map-view arrangement and pathways to discharge points. Hypsometric analysis examines the erosional stages of basins indicated by the hypsometric integrals (HI) and hypsometric curves. A basin and its subbasins show different levels of geologic development that the disaggregation of large basin into small hydrologic units enables the identification of areas of different erosional stages. The prioritization of subbasins considers the intersection of highly mobile divides and highly erosional areas. Over the study area, nine subbasins are identified which are all located at the headwaters of major basins in the island. A considerable earthquake-triggered landslide has been found in one of the identified subbasins. The study presents a new approach in the initial characterization of landforms in order to facilitate the identification and prioritization of highly erodible areas for high consideration especially at the local or village level.

scholarly journals Nucleotides at the extremities of the viral RNA of influenza C virus are involved in type-specific interactions with the polymerase complex

2001 ◽  
Vol 82 (5) ◽  
pp. 1075-1083 ◽  
Author(s):  
Bernadette Crescenzo-Chaigne ◽  
Sylvie van der Werf
Keyword(s):  
Influenza A ◽  
Expression System ◽  
Type A ◽  
Viral Rna ◽  
Noncoding Regions ◽  
Polymerase Complex ◽  
Influenza C Virus ◽  
Type C ◽  
The Stability ◽  
Transcription And Replication

Influenza A and C viruses share common sequences in the terminal noncoding regions of the viral RNA segments. Differences at the 5′- and 3′-ends exist, however, that could contribute to the specificity with which the transcription/replication signals are recognized by the cognate polymerase complexes. Previously, by making use of a transient expression system for the transcription and replication of a reporter RNA template bearing either type A or type C extremities, it was shown that a type C RNA template is transcribed and replicated with equal efficiency by either the type A or the type C polymerase complex, whereas a type A RNA template is less efficiently transcribed and replicated by the type C polymerase complex than by the type A complex. To explore the contribution of the nucleotides at the extremities of the RNAs to this type-specificity, the effect of mutations introduced either alone or in combination at nucleotide 5 at the 3′-end and at nucleotides 3′, 6′ or 8′ at the 5′-end of type A or C RNA templates were studied in the presence of either the type A or the type C polymerase complex. The results indicate that the nature of nucleotides 5 and 6′ contribute to type-specificity. Moreover, these results underline the importance of the base pairing between nucleotide 3′ and 8′ at the 5′-end of the RNA. Thus, it could be suggested that the nature of the nucleotides as well as the stability of the secondary structure at the extremities of the viral RNA are important determinants of type-specificity.

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