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.

scholarly journals Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.

1997 ◽  
Vol 17 (9) ◽  
pp. 5001-5015 ◽  
Author(s):  
N I Zanchin ◽  
P Roberts ◽  
A DeSilva ◽  
F Sherman ◽  
D S Goldfarb
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Open Reading Frames ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Protein Fusion ◽  
Acid Protein ◽  
Conserved Gene ◽  
Green Fluorescent

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.

scholarly journals HPR1 encodes a global positive regulator of transcription in Saccharomyces cerevisiae.

1995 ◽  
Vol 15 (3) ◽  
pp. 1698-1708 ◽  
Author(s):  
Y Zhu ◽  
C L Peterson ◽  
M F Christman
Keyword(s):  
Topoisomerase I ◽  
Gene Dosage ◽  
Normal Growth ◽  
Growth Defect ◽  
Dna Topoisomerase ◽  
Upstream Activation Sequence ◽  
Dam Methylase ◽  
Severe Growth Defect ◽  
Activation Sequence

The Hpr1 protein has an unknown function, although it contains a region of homology to DNA topoisomerase I. We have found that hpr1 null mutants are defective in the transcription of many physiologically unrelated genes, including GAL1, HO, ADH1, and SUC2, by using a combination of Northern (RNA) blot analysis, primer extension, and upstream activation sequence-lacZ fusions. Many of the genes positively regulated by HPR1 also require SWI1, SWI2-SNF2, SWI3, SNF5, and SNF6. The transcriptional defect at HO and the CCB::lacZ upstream activation sequence in hpr1 mutants is partially suppressed by a deletion of SIN1, which encodes an HMG1p-like protein. Elevated gene dosage of either histones H3 and H4 or H2A and H2B results in a severe growth defect in combination with an hpr1 null mutation. However, increased gene dosage of all four histones simultaneously restores near-normal growth in hpr1 mutants. Altered in vivo Dam methylase sensitivity is observed at two HPR1-dependent promoters (GAL1 and SUC2). Most of the Hpr1 protein present in the cell is in a large complex (10(6) Da) that is distinct from the SWI-SNF protein complex. We propose that HPR1 affects transcription and recombination by altering chromatin structure.

Cloning and functional analysis of the dpm2 and dpm3 genes from Trichoderma reesei expressed in a Saccharomyces cerevisiae dpm1Δ mutant strain

2011 ◽  
Vol 392 (6) ◽  
Author(s):  
Patrycja Zembek ◽  
Urszula Perlińska-Lenart ◽  
Katarzyna Rawa ◽  
Wioletta Górka-Nieć ◽  
Grażyna Palamarczyk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Analysis ◽  
Cell Wall ◽  
Enzymatic Activity ◽  
Trichoderma Reesei ◽  
Cell Wall Composition ◽  
Regulatory Subunit ◽  
Dolichyl Phosphate ◽  
Key Enzyme

AbstractInTrichoderma reesei, dolichyl phosphate mannose (dpm) synthase, a key enzyme in the O-glycosylation process, requires three proteins for full activity. In this study, thedpm2anddpm3genes coding for the DPMII and DPMIII subunits ofT. reeseiDPM synthase were cloned and functionally analyzed after expression in theSaccharomyces cerevisiae dpm1Δ[genotype (BY4743;his3Δ1; /leu2Δ0; lys2Δ0; /ura3Δ0; YPR183w::kanMX4] mutant. It was found that apart from the catalytic subunit DPMI, the DPMIII subunit is also essential to form an active DPM synthase in yeast. Additional expression of the DPMII protein, considered to be a regulatory subunit of DPM synthase, decreased the enzymatic activity. We also characterizedS. cerevisiaestrains expressing thedpm1,2,3ordpm1, 3genes and analyzed the consequences ofdpmexpression on protein O-glycosylationin vivoand on the cell wall composition.

scholarly journals bioA mutant of Mycobacterium tuberculosis shows severe growth defect and imparts protection against tuberculosis in guinea pigs

PLoS ONE ◽  
2017 ◽  
Vol 12 (6) ◽  
pp. e0179513 ◽  
Author(s):  
Ritika Kar ◽  
Prachi Nangpal ◽  
Shubhita Mathur ◽  
Swati Singh ◽  
Anil K. Tyagi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Guinea Pigs ◽  
Growth Defect ◽  
Severe Growth Defect ◽  
Severe Growth

scholarly journals Dynamical localization of a thylakoid membrane binding protein is required for acquisition of photosynthetic competency

2017 ◽  
Author(s):  
Andrian Gutu ◽  
Frederick Chang ◽  
Erin K. O‘Shea
Keyword(s):  
Spatial Distribution ◽  
Thylakoid Membrane ◽  
Growth Conditions ◽  
Dynamical Localization ◽  
Growth Defect ◽  
Cell Periphery ◽  
Diffuse Fraction ◽  
Photosynthetic Complexes ◽  
Severe Growth Defect ◽  
Severe Growth

SUMMARYVipp1 is highly conserved and essential for photosynthesis, but its function is unclear as it does not participate directly in light-dependent reactions. We analyzed Vipp1 localization in live cyanobacterial cells and show that Vipp1 is highly dynamic, continuously exchanging between a diffuse fraction that is uniformly distributed throughout the cell and a punctate fraction that is concentrated at high curvature regions of the thylakoid located at the cell periphery. Experimentally perturbing the spatial distribution of Vipp1 by relocalizing it to the nucleoid causes a severe growth defect during the transition from non-photosynthetic (dark) to photosynthetic (light) growth. However, the same perturbation of Vipp1 in dark alone or light alone growth conditions causes no growth or thylakoid morphology defects. We propose that the punctuated dynamics of Vipp1 at the cell periphery in regions of high thylakoid curvature enable acquisition of photosynthetic competency, perhaps by facilitating biogenesis of photosynthetic complexes involved in light-dependent reactions of photosynthesis.

scholarly journals Insufficient levels of thenrdAB-encoded ribonucleotide reductase underlie the severe growth defect of the Δhda E. colistrain

2017 ◽  
Vol 104 (3) ◽  
pp. 377-399 ◽  
Author(s):  
Vignesh M. P. Babu ◽  
Mark Itsko ◽  
Jamie C. Baxter ◽  
Roel M. Schaaper ◽  
Mark D. Sutton
Keyword(s):  
Ribonucleotide Reductase ◽  
Growth Defect ◽  
Severe Growth Defect ◽  
Severe Growth

scholarly journals RelA/p65 Regulation of IκBβ

2005 ◽  
Vol 25 (12) ◽  
pp. 4956-4968 ◽  
Author(s):  
Erin Hertlein ◽  
Jingxin Wang ◽  
Katherine J. Ladner ◽  
Nadine Bakkar ◽  
Denis C. Guttridge
Keyword(s):  
Postnatal Development ◽  
26S Proteasome ◽  
Carboxyl Terminus ◽  
Growth Defect ◽  
Tight Control ◽  
Resistant Form ◽  
Specific Manner ◽  
Severe Growth Defect ◽  
Severe Growth ◽  
Hyperphosphorylated Form

ABSTRACT IκB inhibitor proteins are the primary regulators of NF-κB. In contrast to the defined regulatory interplay between NF-κB and IκBα, much less is known regarding the regulation of IκBβ by NF-κB. Here, we describe in detail the regulation of IκBβ by RelA/p65. Using p65 −/− fibroblasts, we show that IκBβ is profoundly reduced in these cells, but not in other NF-κB subunit knockouts. This regulation prevails during embryonic and postnatal development in a tissue-specific manner. Significantly, in both p65 −/− cells and tissues, IκBα is also reduced, but not nearly to the same extent as IκBβ, thus highlighting the degree to which IκBβ is dependent on p65. This dependence is based on the ability of p65 to stabilize IκBβ protein from the 26S proteasome, a process mediated in large part through the p65 carboxyl terminus. Furthermore, IκBβ was found to exist in both a basally phosphorylated and a hyperphosphorylated form. While the hyperphosphorylated form is less abundant, it is also more stable and less dependent on p65 and its carboxyl domain. Finally, we show that in p65 −/− fibroblasts, expression of a proteolysis-resistant form of IκBβ, but not IκBα, causes a severe growth defect associated with apoptosis. Based on these findings, we propose that tight control of IκBβ protein by p65 is necessary for the maintenance of cellular homeostasis.

scholarly journals The REG2 gene of Saccharomyces cerevisiae encodes a type 1 protein phosphatase-binding protein that functions with Reg1p and the Snf1 protein kinase to regulate growth.

1996 ◽  
Vol 16 (6) ◽  
pp. 2922-2931 ◽  
Author(s):  
D L Frederick ◽  
K Tatchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Protein Phosphatase ◽  
Slow Growth ◽  
Glucose Repression ◽  
Growth Defect ◽  
Loss Of Function ◽  
Cell Extracts

The GLC7 gene of Saccharomyces cerevisiae encodes the catalytic subunit of type 1 protein phosphatase (PP1) and is essential for cell growth. We have isolated a previously uncharacterized gene, REG2, on the basis of its ability to interact with Glc7p in the two-hybrid system. Reg2p interacts with Glc7p in vivo, and epitope-tagged derivatives of Reg2p and Glc7p coimmunoprecipitate from cell extracts. The predicted protein product of the REG2 gene is similar to Reg1p, a protein believed to direct PP1 activity in the glucose repression pathway. Mutants with a deletion of reg1 display a mild slow-growth defect, while reg2 mutants exhibit a wild-type phenotype. However, mutants with deletions of both reg1 and reg2 exhibit a severe growth defect. Overexpression of REG2 complements the slow-growth defect of a reg1 mutant but does not complement defects in glycogen accumulation or glucose repression, two traits also associated with a reg1 deletion. These results indicate that REG1 has a unique role in the glucose repression pathway but acts together with REG2 to regulate some as yet uncharacterized function important for growth. The growth defect of a reg1 reg2 double mutant is alleviated by a loss-of-function mutation in the SNF1-encoded protein kinase. The snf1 mutation also suppresses the glucose repression defects of reg1. Together, our data are consistent with a model in which Reg1p and Reg2p control the activity of PP1 toward substrates that are phosphorylated by the Snf1p kinase.

scholarly journals Human mutation D157G in ferroportin leads to hepcidin-independent binding of Jak2 and ferroportin down-regulation

Blood ◽  
2010 ◽  
Vol 115 (14) ◽  
pp. 2956-2959 ◽  
Author(s):  
Ivana De Domenico ◽  
Eric Lo ◽  
Diane M. Ward ◽  
Jerry Kaplan
Keyword(s):  
Iron Homeostasis ◽  
Fluorescent Protein ◽  
Transferrin Saturation ◽  
High Serum ◽  
Growth Defect ◽  
Down Regulation ◽  
Severe Growth Defect ◽  
Human Mutation ◽  
Green Fluorescent ◽  
Severe Growth

Abstract Mutations in the iron exporter ferroportin (Fpn) result in iron overload in macrophages or hepatocytes depending upon the mutation. Patients with Fpn mutation D157G show high serum ferritin and normal to slightly elevated transferrin saturation. Here, we show that Fpn(D157G)–green fluorescent protein (GFP) is down-regulated independent of hepcidin, and that this down-regulation is due to the constitutive binding of Jak2 and Fpn phosphorylation. Expression of Fpn(D157G)-GFP in Danio rerio results in a severe growth defect, which can be rescued by iron supplementation. These results identify a hepcidin-independent regulation of Fpn that can result in alterations in iron homeostasis.

scholarly journals Saccharomyces cerevisiae RAI1 (YGL246c) Is Homologous to Human DOM3Z and Encodes a Protein That Binds the Nuclear Exoribonuclease Rat1p

2000 ◽  
Vol 20 (11) ◽  
pp. 4006-4015 ◽  
Author(s):  
Yang Xue ◽  
Xinxue Bai ◽  
Insuk Lee ◽  
George Kallstrom ◽  
Jennifer Ho ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Rna Degradation ◽  
Growth Defect ◽  
Nuclear Targeting ◽  
Interacting Protein ◽  
Rrna Species ◽  
Synthetically Lethal

ABSTRACT The RAT1 gene of Saccharomyces cerevisiaeencodes a 5′→3′ exoribonuclease which plays an essential role in yeast RNA degradation and/or processing in the nucleus. We have cloned a previously uncharacterized gene (YGL246c) that we refer to asRAI1 (Rat1p interacting protein 1). RAI1 is homologous to Caenorhabditis elegans DOM-3 and humanDOM3Z. Deletion of RAI1 confers a growth defect which can be complemented by an additional copy of RAT1 on a centromeric vector or by directing Xrn1p, the cytoplasmic homolog of Rat1p, to the nucleus through the addition of a nuclear targeting sequence. Deletion of RAI1 is synthetically lethal with therat1-1ts mutation and shows genetic interaction with a deletion of SKI2 but not XRN1. Polysome analysis of an rai1 deletion mutant indicated a defect in 60S biogenesis which was nearly fully reversed by high-copyRAT1. Northern blot analysis of rRNAs revealed thatrai1 is required for normal 5.8S processing. In the absence of RAI1, 5.8SL was the predominant form of 5.8S and there was an accumulation of 3′-extended forms but not 5′-extended species of 5.8S. In addition, a 27S pre-rRNA species accumulated in therai1 mutant. Thus, deletion of RAI1 affects both 5′ and 3′ processing reactions of 5.8S rRNA. Consistent with the in vivo data suggesting that RAI1 enhances RAT1function, purified Rai1p stabilized the in vitro exoribonuclease activity of Rat1p.

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