Genome-wide screen of Saccharomyces cerevisiae null allele strains identifies genes involved in selenomethionine resistance

Selenomethionine (SeMet) is a potentially toxic amino acid, and yet it is a valuable tool in the preparation of labeled proteins for multiwavelength anomalous dispersion or single-wavelength anomalous dispersion phasing in X-ray crystallography. The mechanism by which high levels of SeMet exhibits its toxic effects in eukaryotic cells is not fully understood. Attempts to use Saccharomyces cerevisiae for the preparation of fully substituted SeMet proteins for X-ray crystallography have been limited. A screen of the viable S. cerevisiae haploid null allele strain collection for resistance to SeMet was performed. Deletion of the CYS3 gene encoding cystathionine gamma-lyase resulted in the highest resistance to SeMet. In addition, deletion of SSN2 resulted in both increased resistance to SeMet as well as reduced levels of Cys3p. A methionine auxotrophic strain lacking CYS3 was able to grow in media with SeMet as the only source of Met, achieving essentially 100% occupancy in total proteins. The CYS3 deletion strain provides advantages for an easy and cost-effective method to prepare SeMet-substituted protein in yeast and perhaps other eukaryotic systems.

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A Simple, Efficient, and Eco-Friendly Method for the Preparation of 3-Substituted-2,3-dihydroquinazolin-4(1H)-one Derivatives

Molecules ◽

10.3390/molecules24224052 ◽

2019 ◽

Vol 24 (22) ◽

pp. 4052 ◽

Cited By ~ 1

Author(s):

Zainab Almarhoon ◽

Kholood A. Dahlous ◽

Rakia Abd Alhameed ◽

Hazem A. Ghabbour ◽

Ayman El-Faham

Keyword(s):

Molecular Structure ◽

Cost Effective ◽

Environmentally Benign ◽

Conventional Heating ◽

Second Step ◽

The Novel ◽

Monoclinic Crystal ◽

X Ray ◽

X Ray Crystallography ◽

Cost Effective Method

A simple, cost-effective method under environmentally benign conditions is a very important concept for the preparation of 2,3-dihydroquinazolin-4(1H)-one derivatives. The present work describes an efficient and eco-friendly protocol for the synthesis of 2-amino-N-(2-substituted-ethyl)benzamide and 3-substituted-2,3-dihydroquinazolin-4(1H)-one derivatives. The novel feature of this protocol is the use of 2-methyl tetrahydrofuran (2-MeTHF) as an eco-friendly alternative solvent to tetrahydrofuran (THF) in the first step. In the second step, methanol in the presence of potassium carbonate as a catalyst was used under conventional heating or microwave irradiation, which provided an eco-friendly method to afford the target products in excellent yields and purities. NMR (1H and 13C), elemental analysis, and LC-MS confirmed the structures of all compounds. X-ray crystallography further confirmed the structure of the intermediate 2-amino-N-(2-substituted-ethyl)benzamide 3a. The molecular structure of 3a was monoclinic crystal, with P21/c, a = 13.6879 (11) Å, b = 10.2118 (9) Å, c = 9.7884 (9) Å, β = 105.068 (7)°, V = 1321.2 (2) Å3, and Z = 4.

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Mutation of High-Affinity Methionine Permease Contributes to Selenomethionyl Protein Production in Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.01026-10 ◽

2010 ◽

Vol 76 (19) ◽

pp. 6351-6359 ◽

Cited By ~ 7

Author(s):

Toshihiko Kitajima ◽

Yasunori Chiba ◽

Yoshifumi Jigami

Keyword(s):

Saccharomyces Cerevisiae ◽

Methylotrophic Yeast ◽

Culture Conditions ◽

Crystallographic Analysis ◽

Gene Encoding ◽

X Ray ◽

X Ray Crystallography ◽

High Affinity ◽

Standard Culture ◽

Resistant Mutants

ABSTRACT The production of selenomethionine (SeMet) derivatives of recombinant proteins allows phase determination by single-wavelength or multiwavelength anomalous dispersion phasing in X-ray crystallography, and this popular approach has permitted the crystal structures of numerous proteins to be determined. Although yeast is an ideal host for the production of large amounts of eukaryotic proteins that require posttranslational modification, the toxic effects of SeMet often interfere with the preparation of protein derivatives containing this compound. We previously isolated a mutant strain (SMR-94) of the methylotrophic yeast Pichia pastoris that is resistant to both SeMet and selenate and demonstrated its applicability for the production of proteins suitable for X-ray crystallographic analysis. However, the molecular basis for resistance to SeMet by the SMR-94 strain remains unclear. Here, we report the characterization of SeMet-resistant mutants of Saccharomyces cerevisiae and the identification of a mutant allele of the MUP1 gene encoding high-affinity methionine permease, which confers SeMet resistance. Although the total methionine uptake by the mup1 mutant (the SRY5-7 strain) decreased to 47% of the wild-type level, it was able to incorporate SeMet into the overexpressed epidermal growth factor peptide with 73% occupancy, indicating the importance of the moderate uptake of SeMet by amino acid permeases other than Mup1p for the alleviation of SeMet toxicity. In addition, under standard culture conditions, the mup1 mutant showed higher productivity of the SeMet derivative relative to other SeMet-resistant mutants. Based on these results, we conclude that the mup1 mutant would be useful for the preparation of selenomethionyl proteins for X-ray crystallography.

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PHF13 is a molecular reader and transcriptional co-regulator of H3K4me2/3

eLife ◽

10.7554/elife.10607 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 14

Author(s):

Ho-Ryun Chung ◽

Chao Xu ◽

Alisa Fuchs ◽

Andreas Mund ◽

Martin Lange ◽

...

Keyword(s):

Dna Damage Response ◽

Size Exclusion ◽

X Ray ◽

Chip Sequencing ◽

X Ray Crystallography ◽

Genome Wide ◽

Damage Response ◽

Exclusion Chromatography ◽

Insight Into ◽

Binding Cell

PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes.

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Detailed differential coefficients of anomalous dispersion terms to derivatives in least-squares refinement for X-ray crystallography

Computers & Chemistry ◽

10.1016/s0097-8485(99)00056-x ◽

2000 ◽

Vol 24 (2) ◽

pp. 143-158 ◽

Cited By ~ 2

Author(s):

Sachiko Okada ◽

Kenji Okada

Keyword(s):

Least Squares ◽

Anomalous Dispersion ◽

X Ray ◽

X Ray Crystallography ◽

Dispersion Terms

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Increased expression of Saccharomyces cerevisiae translation elongation factor 1 alpha bypasses the lethality of a TEF5 null allele encoding elongation factor 1 beta.

Genetics ◽

10.1093/genetics/141.2.481 ◽

1995 ◽

Vol 141 (2) ◽

pp. 481-489 ◽

Cited By ~ 8

Author(s):

T G Kinzy ◽

J L Woolford

Keyword(s):

Saccharomyces Cerevisiae ◽

Null Allele ◽

Elongation Factor ◽

Wild Type ◽

Translation Elongation Factor ◽

Gene Encoding ◽

Translation Elongation ◽

Dominant Mutant ◽

Cold Sensitive ◽

Elongation Factor 1

Abstract Translation elongation factor 1beta (EF-1beta) catalyzes the exchange of bound GDP for GTP on EF-1alpha. The lethality of a null allele of the TEF5 gene encoding EF-1beta in Saccharomyces cerevisiae was suppressed by extra copies of the TEF2 gene encoding EF-1alpha. The strains with tef5::TRP1 suppressed by extra copies of TEF were slow growing, cold sensitive, hypersensitive to inhibitors of translation elongation and showed increased phenotypic suppression of +1 frameshift and UAG nonsense mutations. Nine dominant mutant alleles of TEF2 that cause increased suppression of frameshift mutations also suppressed the lethality of tef5::TRP1. Most of the strains in which tef5::TRP1 is suppressed by dominant mutant alleles of TEF2 grew more slowly and were more antibiotic sensitive than strains with tef5::TRP1 is suppressed by wild-type TEF2. Two alleles, TEF2-4 and TEF2-10, interact with tef5::TRP1 to produce strains that showed doubling times similar to tef5::TRP1 strains containing extra copies of wild-type TEF2. These strains were less cold sensitive, drug sensitive and correspondingly less efficient suppressor of +1 frameshift mutations. These phenotypes indicate that translation and cell growth are highly sensitive to changes in EF-1alpha and EF-1beta activity.

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MAPPING OF LONG-RANGE CHROMATIN INTERACTIONS BY PROXIMITY LIGATION ASSISTED CHIP-SEQ

10.1101/074294 ◽

2016 ◽

Author(s):

Rongxin Fang ◽

Miao Yu ◽

Guoqiang Li ◽

Sora Chee ◽

Tristin Liu ◽

...

Keyword(s):

Chromatin Immunoprecipitation ◽

Embryonic Stem ◽

Cost Effective ◽

Eukaryotic Cells ◽

Proximity Ligation ◽

Chromatin Interactions ◽

Genome Wide ◽

Cost Effective Method ◽

Highly Sensitive ◽

State Of Art

AbstractWe report a highly sensitive and cost-effective method for genome-wide identification of chromatin interactions in eukaryotic cells. Combining proximity ligation with chromatin immunoprecipitation and sequencing, the method outperforms the state of art approach in sensitivity, accuracy and ease of operation. Application of the method to mouse embryonic stem cells improves mapping of enhancer-promoter interactions.

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Mechanical and bioactivity assessment of wollastonite/PVA composite synthesized from bentonite clay

Cerâmica ◽

10.1590/0366-69132019653742584 ◽

2019 ◽

Vol 65 (374) ◽

pp. 246-251

Author(s):

L. A. Adams ◽

E. R. Essien ◽

E. E. Kaufmann

Keyword(s):

Compressive Strength ◽

Bone Regeneration ◽

Biomedical Applications ◽

Bentonite Clay ◽

Cost Effective ◽

Biological Properties ◽

X Ray ◽

Silica Source ◽

Cost Effective Method ◽

Matrix Configuration

Abstract Glass/polymer composites can mimic the natural structure of bone by possessing a fiber-matrix configuration which provides appropriate physical and biological properties. Wollastonite ceramics are known for their promising bioactivity and biocompatibility when applied in bone regeneration. Polyvinyl alcohol (PVA) has various attractive properties including biocompatibility and degradability which may be exploited as a polymer matrix in composites for biomedical applications. Therefore, a cost-effective method of preparing wollastonite/PVA composites is desirable by starting from bentonite clay as a silica source for the glass, instead of traditional alkoxysilanes. The composite prepared was characterized by mechanical testing, scanning electron microscopy, X-ray diffractometry and Fourier-transform infrared spectroscopy to evaluate its compressive strength, morphology, phase composition and bioactivity, respectively. Results obtained revealed for the composite a compressive strength of 0.3 MPa, the ability to induce apatite on its surface when immersed in a simulated body fluid for 7 days and desirable controlled degradation. Hence, this method can be up-scaled for preparation of wollastonite/PVA composite commercially for possible use in bone regeneration.

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