scholarly journals Differential Expression of Individual Suppressor tRNATrp Gene Family Members In Vitro and In Vivo in the Nematode Caenorhabditis elegans

1998 ◽  
Vol 18 (2) ◽  
pp. 703-709 ◽  
Author(s):  
Ling Li ◽  
Rob M. Linning ◽  
Kazunori Kondo ◽  
Barry M. Honda
Keyword(s):  
Caenorhabditis Elegans ◽  
Gene Family ◽  
Differential Expression ◽  
Reporter Gene ◽  
Trna Genes ◽  
Wild Type ◽  
Suppressor Activity ◽  
Cell Extracts

ABSTRACT Eight different amber suppressor tRNA (suptRNA) mutations in the nematode Caenorhabditis elegans have been isolated; all are derived from members of the tRNATrp gene family (K. Kondo, B. Makovec, R. H. Waterston, and J. Hodgkin, J. Mol. Biol. 215:7–19, 1990). Genetic assays of suppressor activity suggested that individual tRNA genes were differentially expressed, probably in a tissue- or developmental stage-specific manner. We have now examined the expression of representative members of this gene family both in vitro, using transcription in embryonic cell extracts, and in vivo, by assaying suppression of an amber-mutated lacZ reporter gene in animals carrying different suptRNA mutations. Individual wild-type tRNATrp genes and their amber-suppressing counterparts appear to be transcribed and processed identically in vitro, suggesting that the behavior of suptRNAs should reflect wild-type tRNA expression. The levels of transcription of different suptRNA genes closely parallel the extent of genetic suppression in vivo. The results suggest that differential expression of tRNA genes is most likely at the transcriptional rather than the posttranscriptional level and that 5′ flanking sequences play a role in vitro, and probably in vivo as well. Using suppression of a lacZ(Am) reporter gene as a more direct assay of suptRNA activity in individual cell types, we have again observed differential expression which correlates with genetic and in vitro transcription results. This provides a model system to more extensively study the basis for differential expression of this tRNA gene family.

scholarly journals Effect of intron mutations on processing and function of Saccharomyces cerevisiae SUP53 tRNA in vitro and in vivo.

1986 ◽  
Vol 6 (7) ◽  
pp. 2663-2673 ◽  
Author(s):  
M C Strobel ◽  
J Abelson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trna Gene ◽  
Nuclear Extract ◽  
Nonsense Suppression ◽  
Trna Genes ◽  
Suppressor Activity ◽  
Specific Sequence ◽  
Made In

The Saccharomyces cerevisiae leucine-inserting amber suppressor tRNA gene SUP53 (a tRNALeu3 allele) was used to investigate the relationship between precursor tRNA structure and mature tRNA function. This gene encodes a pre-tRNA which contains a 32-base intron. The mature tRNASUP53 contains a 5-methylcytosine modification of the anticodon wobble base. Mutations were made in the SUP53 intron. These mutant genes were transcribed in an S. cerevisiae nuclear extract preparation. In this extract, primary tRNA gene transcripts are end-processed and base modified after addition of cofactors. The base modifications made in vitro were examined, and the mutant pre-tRNAs were analyzed for their ability to serve as substrates for partially purified S. cerevisiae tRNA endonuclease and ligase. Finally, the suppressor function of these mutant tRNA genes was assayed after their integration into the S. cerevisiae genome. Mutant analysis showed that the totally intact precursor tRNA, rather than any specific sequence or structure of the intron, was necessary for efficient nonsense suppression by tRNASUP53. Less efficient suppressor activity correlated with the absence of the 5-methylcytosine modification. Most of the intron-altered precursor tRNAs were successfully spliced in vitro, indicating that modifications are not critical for recognition by the tRNA endonuclease and ligase.

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro

1987 ◽  
Vol 7 (10) ◽  
pp. 3694-3704
Author(s):  
C Prives ◽  
Y Murakami ◽  
F G Kern ◽  
W Folk ◽  
C Basilico ◽  
...  
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Early Gene ◽  
Wild Type ◽  
The Core ◽  
Cell Extracts ◽  
Sequence Requirements

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.

Organization of the noncontiguous promoter components of adenovirus VAI RNA gene is strikingly similar to that of eucaryotic tRNA genes

1983 ◽  
Vol 3 (11) ◽  
pp. 1996-2005
Author(s):  
R A Bhat ◽  
B Metz ◽  
B Thimmappaya
Keyword(s):  
Dna Sequences ◽  
Transcriptional Control ◽  
Transcriptional Activity ◽  
Evolutionary Relationship ◽  
Trna Genes ◽  
Wild Type ◽  
Base Pairs ◽  
Internal Promoter ◽  
Cell Extracts

The intragenic transcriptional control region (internal promoter) of the adenovirus type 2 VAI RNA gene was mutated by deletion, insertion, and substitution of DNA sequences at the plasmid level. The mutant plasmids were assayed for in vitro transcriptional activity by using HeLa cell extracts. The mutant clones with substitution or insertion of DNA sequences or both between nucleotides +18 and +53 of the VAI RNA gene were all transcriptionally active, although to various extents. Substitution of unrelated DNA sequences up to +26 or between +54 and +61 abolished the transcriptional activity completely. Based on these results, the intragenic promoter sequences of the VAI RNA gene can be subdivided into two components: element A, +10 to +18; and element B, +54 to +69. The distance between the A and B components could be enlarged from its normal 35 base pairs to 75 base pairs without destroying the transcriptional activity. However, a deletion of 4 or 6 base pairs in the DNA segment separating the A and B components (segment C) reduced the transcriptional activity of the genes to less than 2% of that of the wild type. When the VAI RNA gene with its element A or B was substituted for the corresponding element A or B of the Xenopus laevis tRNAMet gene, the hybrid genes transcribed close to the level of the wild-type VAI RNA gene and about 10- to 20-fold more efficiently than the tRNAMet gene. Thus, the organization of DNA sequences in the internal promoter of the VAI RNA gene appears to be very similar to that of eucaryotic tRNA genes. This similarity suggests an evolutionary relationship of the VAI RNA gene to tRNA genes.

Comparison of Vibrio and Firefly Luciferases as Reporter Gene Systems for Use in Bacteria and Plants

1996 ◽  
Vol 23 (1) ◽  
pp. 75 ◽  
Author(s):  
SR Mudge ◽  
WR Lewis-Henderson ◽  
RG Birch
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Ccd Camera ◽  
Reporter Gene Expression ◽  
In Vitro Assays ◽  
E Coli ◽  
Cell Extracts ◽  
Cooled Ccd

Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

scholarly journals Dual inhibition of MDMX and MDM2 as a therapeutic strategy in leukemia

2018 ◽  
Vol 10 (436) ◽  
pp. eaao3003 ◽  
Author(s):  
Luis A. Carvajal ◽  
Daniela Ben Neriah ◽  
Adrien Senecal ◽  
Lumie Benard ◽  
Victor Thiruthuvanathan ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Single Molecule ◽  
Cellular Proliferation ◽  
Wild Type ◽  
Suppressor Activity ◽  
Mouse Double Minute ◽  
Double Minute ◽  
Wild Type P53

The tumor suppressor p53 is often inactivated via its interaction with endogenous inhibitors mouse double minute 4 homolog (MDM4 or MDMX) or mouse double minute 2 homolog (MDM2), which are frequently overexpressed in patients with acute myeloid leukemia (AML) and other cancers. Pharmacological disruption of both of these interactions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in cancers with wild-type p53. Selective targeting of this pathway has thus far been limited to MDM2-only small-molecule inhibitors, which lack affinity for MDMX. We demonstrate that dual MDMX/MDM2 inhibition with a stapled α-helical peptide (ALRN-6924), which has recently entered phase I clinical testing, produces marked antileukemic effects. ALRN-6924 robustly activates p53-dependent transcription at the single-cell and single-molecule levels and exhibits biochemical and molecular biological on-target activity in leukemia cells in vitro and in vivo. Dual MDMX/MDM2 inhibition by ALRN-6924 inhibits cellular proliferation by inducing cell cycle arrest and apoptosis in cell lines and primary AML patient cells, including leukemic stem cell–enriched populations, and disrupts functional clonogenic and serial replating capacity. Furthermore, ALRN-6924 markedly improves survival in AML xenograft models. Our study provides mechanistic insight to support further testing of ALRN-6924 as a therapeutic approach in AML and other cancers with wild-type p53.

scholarly journals Regulated expression of a mammalian nonsense suppressor tRNA gene in vivo and in vitro using the lac operator/repressor system.

1992 ◽  
Vol 12 (10) ◽  
pp. 4271-4278 ◽  
Author(s):  
D E Syroid ◽  
R I Tapping ◽  
J P Capone
Keyword(s):  
Mammalian Cells ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Lac Repressor ◽  
Trna Genes ◽  
Coding Region ◽  
Lac Operator ◽  
Cell Extracts

We have exploited the Escherichia coli lac operator/repressor system as a means to regulate the expression of a mammalian tRNA gene in vivo and in vitro. An oligonucleotide containing a lac operator (lacO) site was cloned immediately upstream of a human serine amber suppressor (Su+) tRNA gene. Insertion of a single lac repressor binding site at position -1 or -32 relative to the coding region had no effect on the amount of functional tRNA made in vivo, as measured by suppression of a nonsense mutation in the E. coli chloramphenicol acetyltransferase gene following cotransfection of mammalian cells. Inclusion of a plasmid expressing the lac repressor in the transfections resulted in 75 to 98% inhibition of suppression activity of lac operator-linked tRNA genes but had no effect on expression of the wild-type gene. Inhibition could be quantitatively relieved with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG). Similarly, transcription in vitro of lac operator-linked tRNA genes in HeLa cell extracts was repressed in the presence of lac repressor, and this inhibition was reversible with IPTG. These results demonstrate that the bacterial lac operator/repressor system can be used to reversibly control the expression of mammalian genes that are transcribed by RNA polymerase III.

Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo

1988 ◽  
Vol 8 (1) ◽  
pp. 361-370
Author(s):  
S Ganguly ◽  
P A Sharp ◽  
U L RajBhandary
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Wild Type ◽  
Cell Extracts ◽  
Amber Suppressor ◽  
Gene Transcripts ◽  
A Site ◽  
Made In

We describe the results of our studies of expression of a Saccharomyces cerevisiae amber suppressor tRNA(Leu) gene (SUP53) in mammalian cells in vivo and in cell extracts in vitro. Parallel studies were carried out with the wild-type (Su-) tRNA(Leu) gene. Extracts from HeLa or CV1 cells transcribed both tRNA(Leu) genes. The transcripts were processed correctly at the 5' and 3' ends and accurately spliced to produce mature tRNA(Leu). Surprisingly, when the same tRNA(Leu) genes were introduced into CV1 cells, only pre-tRNAs(Leu) were produced. The pre-tRNAs(Leu) made in vivo were of the same size and contained the 5'-leader and 3'-trailer sequences as did pre-tRNAs(Leu) made in vitro. Furthermore, the pre-tRNAs(Leu) made in vivo were processed to mature tRNA(Leu) when incubated with HeLa cell extracts. A tRNA(Leu) gene from which the intervening sequence had been removed yielded RNAs that also were not processed at either their 5' or 3' termini. Thus, processing of pre-tRNA(Leu) in CV1 cells is blocked at the level of 5'- and 3'-end maturation. One possible explanation of the discrepancy in the results obtained in vivo and in vitro is that tRNA biosynthesis in mammalian cells involves transport of pre-tRNA from the site of its synthesis to a site or sites where processing takes place, and perhaps the yeast pre-tRNAs(Leu) synthesized in CV1 cells are not transported to the appropriate site.

scholarly journals Comparative phytochemical analysis of Phlogacanthus thyrsiflorus Nees: Implications on attenuation of pro-oxidants and pathogen virulence in Caenorhabditis elegans model system

2017 ◽  
Vol 10 (5) ◽  
pp. 361
Author(s):  
Kitlangki Suchiang ◽  
Nitasha H Kayde
Keyword(s):  
Oxidative Stress ◽  
Free Radicals ◽  
Free Radical ◽  
Caenorhabditis Elegans ◽  
Model System ◽  
Wild Type ◽  
C Elegans ◽  
Mutant Strains

Background: Phlogacanthus thyrsiflorus Nees (P. thyrsiflorus) of Acanthaceae family is endogenous to sub-tropical Himalayas. It has been reported to be used traditionally in Jaintia tribe of Meghalaya, India for treatment of many ailments.Objectives: The aim was to detect the active compounds present in the leaves for evaluation of in vitro free radicals scavenging potentials. Leaves protective actions in vivo will be investigated using Caenorhabditis elegans (C. elegans) model system utilizing wild type and mutant strains and the phenomena of host-pathogens interactions.Materials and methods: Gas chromatography/ Mass spectrometry (GC/MS) was used for detection of different compounds present. The versatility of leaf extracts to scavenge different free radicals generated in vitro was assessed with different in vitro methods. Survival analysis of wild type and mutant strains C. elegans under enhanced pro-oxidants exposure was investigated in vivo. Fast killing assay was also performed to study the extracts modulatory activity on host C. elegans survival under pathogen Pseudomonas aeruginosa infection.Results:  Forty compounds were detected in methanolic fraction of the extract with variable percentages. Both aqueous and methanol extract possessed remarkable, versatile free radical scavenging activity irrespective of the types of free radical generated. The in vivo experiments are in compliance, with observable increased survival ability percentage of C. elegans under intense exogenous oxidative stress and pathogen infection.Conclusion: Our findings enlightened the different compounds present with versatility of P. thyrsiflorus in tackling different free radicals generated both in vitro and in vivo that highly support for its candidature as a good antioxidant source. Our findings may justify the historical relevance of this plant in herbal remedies that could form the basis for inquiry of new active principles.Keywords: Free radicals, Oxidative stress, Caenorhabditis elegans, Phlogacanthus thyrsiflorus, Phytochemicals

scholarly journals Organization of the noncontiguous promoter components of adenovirus VAI RNA gene is strikingly similar to that of eucaryotic tRNA genes.

1983 ◽  
Vol 3 (11) ◽  
pp. 1996-2005 ◽  
Author(s):  
R A Bhat ◽  
B Metz ◽  
B Thimmappaya
Keyword(s):  
Dna Sequences ◽  
Transcriptional Control ◽  
Transcriptional Activity ◽  
Evolutionary Relationship ◽  
Trna Genes ◽  
Wild Type ◽  
Base Pairs ◽  
Internal Promoter ◽  
Cell Extracts

The intragenic transcriptional control region (internal promoter) of the adenovirus type 2 VAI RNA gene was mutated by deletion, insertion, and substitution of DNA sequences at the plasmid level. The mutant plasmids were assayed for in vitro transcriptional activity by using HeLa cell extracts. The mutant clones with substitution or insertion of DNA sequences or both between nucleotides +18 and +53 of the VAI RNA gene were all transcriptionally active, although to various extents. Substitution of unrelated DNA sequences up to +26 or between +54 and +61 abolished the transcriptional activity completely. Based on these results, the intragenic promoter sequences of the VAI RNA gene can be subdivided into two components: element A, +10 to +18; and element B, +54 to +69. The distance between the A and B components could be enlarged from its normal 35 base pairs to 75 base pairs without destroying the transcriptional activity. However, a deletion of 4 or 6 base pairs in the DNA segment separating the A and B components (segment C) reduced the transcriptional activity of the genes to less than 2% of that of the wild type. When the VAI RNA gene with its element A or B was substituted for the corresponding element A or B of the Xenopus laevis tRNAMet gene, the hybrid genes transcribed close to the level of the wild-type VAI RNA gene and about 10- to 20-fold more efficiently than the tRNAMet gene. Thus, the organization of DNA sequences in the internal promoter of the VAI RNA gene appears to be very similar to that of eucaryotic tRNA genes. This similarity suggests an evolutionary relationship of the VAI RNA gene to tRNA genes.

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