scholarly journals In vivo identification of essential nucleotides in tRNA Leu to its functions by using a constructed yeast tRNA Leu knockout strain

2012 ◽  
Vol 40 (20) ◽  
pp. 10463-10477 ◽  
Author(s):  
Qian Huang ◽  
Peng Yao ◽  
Gilbert Eriani ◽  
En-Duo Wang
Keyword(s):  
Knockout Strain ◽  
Yeast Trna

scholarly journals Trm11p and Trm112p Are both Required for the Formation of 2-Methylguanosine at Position 10 in Yeast tRNA

2005 ◽  
Vol 25 (11) ◽  
pp. 4359-4370 ◽  
Author(s):  
Suresh K. Purushothaman ◽  
Janusz M. Bujnicki ◽  
Henri Grosjean ◽  
Bruno Lapeyre
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzymatic Activity ◽  
Zinc Binding ◽  
Growth Defect ◽  
Putative Protein ◽  
Yeast Trna ◽  
Protein Methyltransferase ◽  
Severe Growth Defect ◽  
Severe Growth

ABSTRACT N 2 -Monomethylguanosine-10 (m2G10) and N 2 ,N 2 -dimethylguanosine-26 (m2 2G26) are the only two guanosine modifications that have been detected in tRNA from nearly all archaea and eukaryotes but not in bacteria. In Saccharomyces cerevisiae, formation of m2 2G26 is catalyzed by Trm1p, and we report here the identification of the enzymatic activity that catalyzes the formation of m2G10 in yeast tRNA. It is composed of at least two subunits that are associated in vivo: Trm11p (Yol124c), which is the catalytic subunit, and Trm112p (Ynr046w), a putative zinc-binding protein. While deletion of TRM11 has no detectable phenotype under laboratory conditions, deletion of TRM112 leads to a severe growth defect, suggesting that it has additional functions in the cell. Indeed, Trm112p is associated with at least four proteins: two tRNA methyltransferases (Trm9p and Trm11p), one putative protein methyltransferase (Mtc6p/Ydr140w), and one protein with a Rossmann fold dehydrogenase domain (Lys9p/Ynr050c). In addition, TRM11 interacts genetically with TRM1, thus suggesting that the absence of m2G10 and m2 2G26 affects tRNA metabolism or functioning.

Turnover of the 5′-end phosphate in yeast tRNA in vivo

FEBS Letters ◽  
1978 ◽  
Vol 89 (2) ◽  
pp. 260-262
Author(s):  
T. Wasiak ◽  
M. Gniazdowski
Keyword(s):  
Yeast Trna

scholarly journals In VivoTitration of Folate Pathway Enzymes

2018 ◽  
Vol 84 (19) ◽  
Author(s):  
Deepika Nambiar ◽  
Timkhite-Kulu Berhane ◽  
Robert Shew ◽  
Bryan Schwarz ◽  
Michael R. Duff ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Dihydrofolate Reductase ◽  
Environmental Changes ◽  
Stress Conditions ◽  
Antibacterial Drug ◽  
Content Type ◽  
E Coli ◽  
Knockout Strain ◽  
R67 Dihydrofolate Reductase

ABSTRACTHow enzymes behave in cells is likely different from how they behave in the test tube. Previousin vitrostudies find that osmolytes interact weakly with folate. Removal of the osmolyte from the solvation shell of folate is more difficult than removal of water, which weakens binding of folate to its enzyme partners. To examine if this phenomenon occursin vivo, osmotic stress titrations were performed withEscherichia coli. Two strategies were employed: resistance to an antibacterial drug and complementation of a knockout strain by the appropriate gene cloned into a plasmid that allows tight control of expression levels as well as labeling by a degradation tag. The abilities of the knockout and complemented strains to grow under osmotic stress were compared. Typically, the knockout strain could grow to high osmolalities on supplemented medium, while the complemented strain stopped growing at lower osmolalities on minimal medium. This pattern was observed for an R67 dihydrofolate reductase clone rescuing a ΔfolAstrain, for a methylenetetrahydrofolate reductase clone rescuing a ΔmetFstrain, and for a serine hydroxymethyltransferase clone rescuing a ΔglyAstrain. Additionally, an R67 dihydrofolate reductase clone allowedE. coliDH5α to grow in the presence of trimethoprim until an osmolality of ∼0.81 is reached, while cells in a control titration lacking antibiotic could grow to 1.90 osmol.IMPORTANCEE. colican survive in drought and flooding conditions and can tolerate large changes in osmolality. However, the cell processes that limit bacterial growth under high osmotic stress conditions are not known. In this study, the dose of four different enzymes inE. coliwas decreased by using deletion strains complemented by the gene carried in a tunable plasmid. Under conditions of limiting enzyme concentration (lower than that achieved by chromosomal gene expression), cell growth can be blocked by osmotic stress conditions that are normally tolerated. These observations indicate thatE. colihas evolved to deal with variations in its osmotic environment and that normal protein levels are sufficient to buffer the cell from environmental changes. Additional factors involved in the osmotic pressure response may include altered protein concentration/activity levels, weak solute interactions with ligands which can make it more difficult for proteins to bind their substrates/inhibitors/cofactorsin vivo, and/or viscosity effects.

scholarly journals Superantigens Subvert the Neutrophil Response To Promote Abscess Formation and Enhance Staphylococcus aureus SurvivalIn Vivo

2014 ◽  
Vol 82 (9) ◽  
pp. 3588-3598 ◽  
Author(s):  
Stacey X. Xu ◽  
Kevin J. Gilmore ◽  
Peter A. Szabo ◽  
Joseph J. Zeppa ◽  
Miren L. Baroja ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Bacterial Pathogen ◽  
Staphylococcal Infection ◽  
Infection Process ◽  
Interleukin 12 ◽  
Bacterial Survival ◽  
Content Type ◽  
Knockout Strain ◽  
Neutrophil Response

ABSTRACTStaphylococcus aureusis a versatile bacterial pathogen that produces T cell-activating toxins known as superantigens (SAgs). Although excessive immune activation by SAgs can induce a dysregulated cytokine storm as a component of what is known as toxic shock syndrome (TSS), the contribution of SAgs to the staphylococcal infection process is not well defined. Here, we evaluated the role of the bacterial superantigen staphylococcal enterotoxin A (SEA) in a bacteremia model using humanized transgenic mice expressing SAg-responsive HLA-DR4 molecules. Infection withS. aureusNewman induced SEA-dependent Vβ skewing of T cells and enhanced bacterial survival in the liver compared with infection byseaknockout strain. SEA-induced gamma interferon, interleukin-12, and chemokine responses resulted in increased infiltration of CD11b+Ly6G+neutrophils into the liver, promoting the formation of abscesses that contained large numbers of viable staphylococci. Hepatic abscesses occurred significantly more frequently inS. aureusNewman-infected livers than in livers infected with the Newmanseaknockout strain, promoting the survival ofS. aureusin vivo. This represents a novel mechanism during infection wherebyS. aureusutilizes SAgs to form a specialized niche and manipulate the immune system.

In vivo modulation of yeast tRNA gene expression by 5′-flanking sequences.

1985 ◽  
Vol 4 (10) ◽  
pp. 2649-2656 ◽  
Author(s):  
K.C. Raymond ◽  
G.J. Raymond ◽  
J.D. Johnson
Keyword(s):  
Gene Expression ◽  
Trna Gene ◽  
Yeast Trna ◽  
Flanking Sequences

scholarly journals A Mycobacterium tuberculosis Rpf Double-Knockout Strain Exhibits Profound Defects in Reactivation from Chronic Tuberculosis and Innate Immunity Phenotypes

2008 ◽  
Vol 76 (9) ◽  
pp. 4269-4281 ◽  
Author(s):  
Eleanor Russell-Goldman ◽  
Jiayong Xu ◽  
Xiaobing Wang ◽  
John Chan ◽  
JoAnn M. Tufariello
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Axenic Culture ◽  
Colony Morphology ◽  
Double Knockout ◽  
Drug Induced ◽  
Necrosis Factor Alpha ◽  
Knockout Strain ◽  
Primary Mouse

ABSTRACT Resuscitation-promoting factors (Rpfs), apparent peptidoglycan hydrolases, have been implicated in the reactivation of dormant bacteria. We previously demonstrated that deletion of rpfB impaired reactivation of Mycobacterium tuberculosis in a mouse model. Because M. tuberculosis encodes five Rpf paralogues, redundant functions among the family members might obscure rpf single-knockout phenotypes. A series of rpf double knockouts were therefore generated. One double mutant, ΔrpfAB, exhibited several striking phenotypes. Consistent with the proposed cell wall-modifying function of Rpfs, ΔrpfAB exhibited an altered colony morphology. Although ΔrpfAB grew comparably to the parental strain in axenic culture, in vivo it exhibited deficiency in reactivation induced in C57BL/6 mice by the administration of nitric oxide synthase inhibitor (aminoguanidine) or by CD4+ T-cell depletion. Notably, the reactivation deficiency of ΔrpfAB was more severe than that of ΔrpfB in aminoguanidine-treated mice. A similar deficiency was observed in ΔrpfAB reactivation from a drug-induced apparently sterile state in infected NOS2−/− mice upon cessation of antimycobacterial therapy. Secondly, ΔrpfAB showed a persistence defect not seen with the ΔrpfB or ΔrpfA single mutants. Interestingly, ΔrpfAB exhibited impaired growth in primary mouse macrophages and induced higher levels of the proinflammatory cytokines tumor necrosis factor alpha and interleukin 6. Simultaneous reintroduction of rpfA and rpfB into the double-knockout strain complemented the colony morphology and macrophage cytokine secretion phenotypes. Phenotypes related to cell wall composition and macrophage responses suggest that M. tuberculosis Rpfs may influence the outcome of reactivation, in part, by modulating innate immune responses to the bacterium.

scholarly journals Requirement of Toxoplasma gondii metacaspases for IMC1 maturation, endodyogeny and virulence in mice

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Muzi Li ◽  
Jing Liu ◽  
Yayun Wu ◽  
Yihan Wu ◽  
Xiaodong Sun ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Normal Growth ◽  
Double Knockout ◽  
Protein Aggregate ◽  
Knockout Strain ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
Single Knockout

Abstract Background Metacaspases are multifunctional proteins found in plants, fungi and protozoa, and are involved in processes such as insoluble protein aggregate clearance and cell proliferation. Our previous study demonstrated that metacaspase-1 (MCA1) contributes to parasite apoptosis in Toxoplasma gondii. Deletion of MCA1 from T. gondii has no effect on the growth and virulence of the parasites. Three metacaspases were identified in the ToxoDB Toxoplasma Informatics Resource, and the function of metacaspase-2 (MCA2) and metacaspase-3 (MCA3) has not been demonstrated. Methods In this study, we constructed MCA1, MCA2 and MCA1/MCA2 transgenic strains from RHΔku80 (Δku80), including overexpressing strains and knockout strains, to clarify the function of MCA1 and MCA2 of T. gondii. Results MCA1 and MCA2 were distributed in the cytoplasm with punctuated aggregation, and part of the punctuated aggregation of MCA1 and MCA2 was localized on the inner membrane complex of T. gondii. The proliferation of the MCA1/MCA2 double-knockout strain was significantly reduced; however, the two single knockout strains (MCA1 knockout strain and MCA2 knockout strain) exhibited normal growth rates as compared to the parental strain, Δku80. In addition, endodyogeny was impaired in the tachyzoites whose MCA1 and MCA2 were both deleted due to multiple nuclei and abnormal expression of IMC1. We further found that IMC1 of the double-knockout strain was detergent-soluble, indicating that MCA1 and MCA2 are associated with IMC1 maturation. Compared to the parental Δku80 strain, the double-knockout strain was more readily induced from tachyzoites to bradyzoites in vitro. Furthermore, the double-knockout strain was less pathogenic in mice and was able to develop bradyzoites in the brain, which formed cysts and established chronic infection. Conclusion MCA1 and MCA2 are important factors which participate in IMC1 maturation and endodyogeny of T. gondii. The double-knockout strain has slower proliferation and was able to develop bradyzoites both in vitro and in vivo. Graphic abstract

scholarly journals A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteins in vivo

2005 ◽  
Vol 102 (46) ◽  
pp. 16729-16734 ◽  
Author(s):  
F. Vignols ◽  
C. Brehelin ◽  
Y. Surdin-Kerjan ◽  
D. Thomas ◽  
Y. Meyer
Keyword(s):  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Knockout Strain ◽  
Two Hybrid

scholarly journals RNA and the Goldilocks Zone: Where Mg2+ concentration is just right

2021 ◽  
Author(s):  
Rebecca Guth-Metzler ◽  
Ahmad Mohyeldin Mohamed ◽  
Elizabeth T Cowan ◽  
Moran Frenkel-Pinter ◽  
Roger Wartell ◽  
...  
Keyword(s):  
Rna Folding ◽  
Local Maximum ◽  
Rna Cleavage ◽  
Early Earth ◽  
Yeast Trna ◽  
Earth Environment ◽  
Evolutionary Context ◽  
Simulation And Experiment ◽  
Early Earth Environment

Mg2+, the most abundant divalent cation in cells, catalyzes RNA cleavage but can also promote RNA folding. Because folding can protect RNA from cleavage, we predicted a "Goldilocks zone", which is a local maximum in RNA lifetime at the minimum Mg2+ concentration required for folding. By simulation and experiment, we characterized the RNA Goldilocks zone and its dependence on cleavage parameters and extent of folding. We show experimentally that yeast tRNAPhe can inhabit a Goldilocks zone. The Goldilocks phenomena appears to be robust and is tunable by changes in magnesium affinity, and a variety of other factors. Goldilocks behavior can be more pronounced for RNAs with intermediate folding states. Goldilocks behavior allows ultrafine control of RNA chemical lifetime. A subset of RNAs in vivo are expected to occupy the Goldilocks zone. In evolutionary context, Goldilocks behavior may have shaped RNA in an early Earth environment containing Mg2+ and other metals. RNAs that do not fold cannot access a Goldilocks zone.

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