Silver Staining of Ribosomal Proteins

1971 ◽  
Vol 9 (1) ◽  
pp. 253-269
Author(s):  
J. W. SMITH ◽  
R. J. STUART
Keyword(s):  
Silver Nitrate ◽  
Perchloric Acid ◽  
Silver Staining ◽  
Ribosomal Proteins ◽  
Nuclear Staining ◽  
Rna Molecules ◽  
Mitochondrial Ribosomes ◽  
Associated Proteins ◽  
Relationship Of ◽  
Formalin Fixed

The effects of staining several tissues with silver nitrate were studied in the electron microscope. Tissues were fixed in 2% glutaraldehyde buffered to pH 7.3 and containing 0.22 M sucrose, some being post-osmicated. Staining was effected by immersion of Araldite sections in unbuffered 5% silver nitrate for 30 min. Increase in electron density was restricted to all nucleic acid-containing structures except mitochondrial DNA which is probably not associated with histones. Treatment of fixed tissues with cold 10% perchloric acid for 24 h, which extracts 85% of the total cell RNA, abolished silver staining within the nucleolus but did not affect that of cytoplasmic or mitochondrial ribosomes. Incubation of isolated, formalin-fixed liver cells with DNase I did not abolish nuclear staining. This evidence suggests that silver nitrate stains selectively the proteins associated with nucleic acids: the abolition of nucleoler staining by perchloric acid is not at present understood but may be due to some difference in relationship of protein to RNA in this, compared with other situations. Silver staining indicates that the loci occupied by RNA-associated proteins differ in size and number in different type of ribosomes. Cytoplasmic ribosomes probably contain 5 loci of 4-6 nm diameter, 2 being situated in the 60-S subunit and 3 in the 40-S subunit. Mitochondrial ribosomes appear to contain 2 smaller loci. In the nucleolus the majority of the ribosomes constituting the granulosa contain 2 unequal loci of about 4 and 6 nm respectively, while the appearance of the pars fibrosa is compatible with the view that it consists of a network of long, extended RNA molecules with which small 2.5-nm protein loci are associated.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
Peptidyl Transferase ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
In Vitro Reconstitution

AbstractRibosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

scholarly journals Comprehensive Evaluation of PAXgene Fixation on Oral Cancer Tissues Using Routine Histology, Immunohistochemistry, and FTIR Microspectroscopy

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 889
Author(s):  
Pooja Lahiri ◽  
Suranjana Mukherjee ◽  
Biswajoy Ghosh ◽  
Debnath Das ◽  
Basudev Lahiri ◽  
...  
Keyword(s):  
Comprehensive Evaluation ◽  
Antigen Retrieval ◽  
Tissue Fixation ◽  
Ftir Microspectroscopy ◽  
Protein Secondary Structures ◽  
Associated Proteins ◽  
Cancer Tissues ◽  
Formalin Fixed ◽  
Fixation System

The choice of tissue fixation is critical for preserving the morphology and biochemical information of tissues. Fragile oral tissues with lower tensile strength are challenging to process for histological applications as they are prone to processing damage, such as tissue tear, wrinkling, and tissue fall-off from slides. This leads to loss of morphological information and unnecessary delay in experimentation. In this study, we have characterized the new PAXgene tissue fixation system on oral buccal mucosal tissue of cancerous and normal pathology for routine histological and immunohistochemical applications. We aimed to minimize the processing damage of tissues and improve the quality of histological experiments. We also examined the preservation of biomolecules by PAXgene fixation using FTIR microspectroscopy. Our results demonstrate that the PAXgene-fixed tissues showed significantly less tissue fall-off from slides. Hematoxylin and Eosin staining showed comparable morphology between formalin-fixed and PAXgene-fixed tissues. Good quality and slightly superior immunostaining for cancer-associated proteins p53 and CK5/6 were observed in PAXgene-fixed tissues without antigen retrieval than formalin-fixed tissues. Further, FTIR measurements revealed superior preservation of glycogen, fatty acids, and amide III protein secondary structures in PAXgene-fixed tissues. Overall, we present the first comprehensive evaluation of the PAXgene tissue fixation system in oral tissues. This study concludes that the PAXgene tissue fixation system can be applied to oral tissues to perform diagnostic molecular pathology experiments without compromising the quality of the morphology or biochemistry of biomolecules.

The effects of spectinomycin and ethidium bromide on the synthesis of organelle rRNA and on ultrastructure in Ochromonas danica

1980 ◽  
Vol 43 (1) ◽  
pp. 119-136
Author(s):  
H. Smith-Johannsen ◽  
D. Fromson ◽  
S.P. Gibbs
Keyword(s):  
Electron Microscopy ◽  
Ethidium Bromide ◽  
Ribosomal Proteins ◽  
Specific Inhibitor ◽  
Chloroplast Ultrastructure ◽  
Normal Amount ◽  
Chloroplast Membranes ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Rrna ◽  
Ochromonas Danica

The effects of 24-h exposure to spectinomycin (100 microgram/ml) and ethidium bromide (1 microgram/ml) on the accumulation of chloroplast and mitochondrial rRNAs and on organelle ultrastructure were studied in greening cells of Ochromonas danica. Cells treated with ethidium bromide for 24 h divide at the same rate as controls but contain less than one third the normal amount of mitochondrial rRNA. Ultrastructural observations showed that these cells contain only 10% the number of mitochondrial ribosomes found in controls as well as fewer mitochondrial cristae. Ethidium bromide has no effect on chloroplast ultrastructure in Ochromonas. Greening cells treated with spectinomycin grow at close to control rates but contain 30–40% less chloroplast rRNA than do controls. Electron microscopy showed that spectinomycin disrupts the organization of chloroplast membranes and reduces the number of chloroplast ribosomes by 30%. Under these conditions, spectinomycin has no effect on mitochondrial rRNA or ultrastructure. Since spectinomycin is a specific inhibitor of translation on 70S ribosomes, these results are consistent with the possibility that at least some chloroplast ribosomal proteins are synthesized in the chloroplast of Ochromonas.

scholarly journals Anatomical Variations of the Recurrent Laryngeal Nerve and Implications for Injury Prevention during Surgical Procedures of the Neck

Diagnostics ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 670
Author(s):  
Alison M. Thomas ◽  
Daniel K. Fahim ◽  
Jickssa M. Gemechu
Keyword(s):  
Recurrent Laryngeal Nerve ◽  
Surgical Procedures ◽  
Anatomical Variations ◽  
Laryngeal Nerve ◽  
Inferior Thyroid Artery ◽  
Branching Pattern ◽  
The Right ◽  
Relationship Of ◽  
Intrinsic Laryngeal Muscles ◽  
Formalin Fixed

Accurate knowledge of anatomical variations of the recurrent laryngeal nerve (RLN) provides information to prevent inadvertent intraoperative injury and ultimately guide best clinical and surgical practices. The present study aims to assess the potential anatomical variability of RLN pertaining to its course, branching pattern, and relationship to the inferior thyroid artery, which makes it vulnerable during surgical procedures of the neck. Fifty-five formalin-fixed cadavers were carefully dissected and examined, with the course of the RLN carefully evaluated and documented bilaterally. Our findings indicate that extra-laryngeal branches coming off the RLN on both the right and left side innervate the esophagus, trachea, and mainly intrinsic laryngeal muscles. On the right side, 89.1% of the cadavers demonstrated 2–5 extra-laryngeal branches. On the left, 74.6% of the cadavers demonstrated 2–3 extra-laryngeal branches. In relation to the inferior thyroid artery (ITA), 67.9% of right RLNs were located anteriorly, while 32.1% were located posteriorly. On the other hand, 32.1% of left RLNs were anterior to the ITA, while 67.9% were related posteriorly. On both sides, 3–5% of RLN crossed in between the branches of the ITA. Anatomical consideration of the variations in the course, branching pattern, and relationship of the RLNs is essential to minimize complications associated with surgical procedures of the neck, especially thyroidectomy and anterior cervical discectomy and fusion (ACDF) surgery. The information gained in this study emphasizes the need to preferentially utilize left-sided approaches for ACDF surgery whenever possible.

scholarly journals Tagging of RPS9 as a tool for ribosome purification and identification of ribosome-associated proteins

2020 ◽  
pp. 57-57
Author(s):  
Bogdan Jovanovic ◽  
Lisa Schubert ◽  
Fabian Poetz ◽  
Georg Stoecklin
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Proteins ◽  
Associated Factors ◽  
Ribosomal Subunit ◽  
High Specificity ◽  
Negative Control ◽  
Tev Protease ◽  
Protease Cleavage ◽  
Associated Proteins ◽  
And Function

Ribosomes, the catalytic machinery required for protein synthesis, are comprised of 4 ribosomal RNAs and about 80 ribosomal proteins in mammals. Ribosomes further interact with numerous associated factors that regulate their biogenesis and function. As mutations of ribosomal proteins and ribosome associated proteins cause many diseases, it is important to develop tools by which ribosomes can be purified efficiently and with high specificity. Here, we designed a method to purify ribosomes from human cell lines by C-terminally tagging human RPS9, a protein of the small ribosomal subunit. The tag consists of a flag peptide and a streptavidin-binding peptide (SBP) separated by the tobacco etch virus (TEV) protease cleavage site. We demonstrate that RPS9-Flag-TEV-SBP (FTS) is efficiently incorporated into the ribosome without interfering with regular protein synthesis. Using HeLa-GFP-G3BP1 cells stably expressing RPS9-FTS or, as a negative control, mCherry-FTS, we show that complete ribosomes as well as numerous ribosome-associated proteins are efficiently and specifically purified following pull-down of RPS9-FTS using streptavidin beads. This tool will be helpful for the characterization of human ribosome heterogeneity, post-translational modifications of ribosomal proteins, and changes in ribosome-associated factors after exposing human cells to different stimuli and conditions.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Large Subunit ◽  
Structural Basis ◽  
Catalytic Core ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
Translation Systems

Ribosome biogenesis is an essential process that requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. In particular, maturation of the peptidyl transferase center (PTC), the catalytic core of the ribosome, is mediated by universally conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial ribosomal large subunit (mtLSU) using a combination of endogenous complex purification, in vitro reconstitution and cryo-electron microscopy (cryo-EM). Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Subsequent addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch by releasing MTERF4-NSUN4 and GTPBP5 accompanied by the progression to a near-mature PTC state. In addition, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results define the molecular basis of dynamic GTPase-mediated PTC maturation during mitochondrial ribosome biogenesis and provide a framework for understanding step-wise progression of PTC folding as a critical quality control checkpoint in all translation systems.

The structure of SAV1646 fromStaphylococcus aureusbelonging to a new `ribosome-associated' subfamily of bacterial proteins

2015 ◽  
Vol 71 (2) ◽  
pp. 332-337 ◽  
Author(s):  
Yuri N. Chirgadze ◽  
Teresa E. Clarke ◽  
Vladimir Romanov ◽  
Gera Kisselman ◽  
Jean Wu-Brown ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Rotation Axis ◽  
Genomic Structure ◽  
Large Subunit ◽  
Functional Model ◽  
Homology Search ◽  
Bacterial Proteins ◽  
Bacterial Ribosome ◽  
Acid Protein ◽  
Associated Proteins

The crystal structure of the SAV1646 protein from the pathogenic microorganismStaphylococcus aureushas been determined at 1.7 Å resolution. The 106-amino-acid protein forms a two-layer sandwich with α/β topology. The protein molecules associate as dimers in the crystal and in solution, with the monomers related by a pseudo-twofold rotation axis. A sequence-homology search identified the protein as a member of a new subfamily of yet uncharacterized bacterial `ribosome-associated' proteins with at least 13 members to date. A detailed analysis of the crystal protein structure along with the genomic structure of the operon containing thesav1646gene allowed a tentative functional model of this protein to be proposed. The SAV1646 dimer is assumed to form a complex with ribosomal proteins L21 and L27 which could help to complete the assembly of the large subunit of the ribosome.

scholarly journals Detecting Protein-Protein Interactions in the Intact Cell of Bacillus subtilis (ATCC 6633)

2003 ◽  
Vol 185 (14) ◽  
pp. 4268-4275 ◽  
Author(s):  
Michael S. Winters ◽  
R. A. Day
Keyword(s):  
Bacillus Subtilis ◽  
Protein Interactions ◽  
Ribosomal Proteins ◽  
Intact Cell ◽  
Protein Biosynthesis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Database ◽  
Haloarcula Marismortui ◽  
Associated Proteins ◽  
Covalently Linked

ABSTRACT The salt bridge, paired group-specific reagent cyanogen (ethanedinitrile; C2N2) converts naturally occurring pairs of functional groups into covalently linked products. Cyanogen readily permeates cell walls and membranes. When the paired groups are shared between associated proteins, isolation of the covalently linked proteins allows their identity to be assigned. Examination of organisms of known genome sequence permits identification of the linked proteins by mass spectrometric techniques applied to peptides derived from them. The cyanogen-linked proteins were isolated by polyacrylamide gel electrophoresis. Digestion of the isolated proteins with proteases of known specificity afforded sets of peptides that could be analyzed by mass spectrometry. These data were compared with those derived theoretically from the Swiss Protein Database by computer-based comparisons (Protein Prospector; http://prospector.ucsf.edu ). Identification of associated proteins in the ribosome of Bacillus subtilis strain ATCC 6633 showed that there is an association homology with the association patterns of the ribosomal proteins of Haloarcula marismortui and Thermus thermophilus. In addition, other proteins involved in protein biosynthesis were shown to be associated with ribosomal proteins.

scholarly journals Gene Expression Profiling of Corpus luteum Reveals Important Insights about Early Pregnancy in Domestic Sheep

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 415 ◽  
Author(s):  
Kisun Pokharel ◽  
Jaana Peippo ◽  
Melak Weldenegodguad ◽  
Mervi Honkatukia ◽  
Meng-Hua Li ◽  
...  
Keyword(s):  
Corpus Luteum ◽  
Early Pregnancy ◽  
Ribosomal Proteins ◽  
Embryo Implantation ◽  
Differentially Expressed ◽  
Domestic Sheep ◽  
Preimplantation Stage ◽  
Sialic Acid Binding ◽  
Associated Proteins ◽  
First Time

The majority of pregnancy loss in ruminants occurs during the preimplantation stage, which is thus the most critical period determining reproductive success. Here, we performed a comparative transcriptome study by sequencing total mRNA from corpus luteum (CL) collected during the preimplantation stage of pregnancy in Finnsheep, Texel and F1 crosses. A total of 21,287 genes were expressed in our data. Highly expressed autosomal genes in the CL were associated with biological processes such as progesterone formation (STAR, CYP11A1, and HSD3B1) and embryo implantation (e.g., TIMP1, TIMP2 and TCTP). Among the list of differentially expressed genes, sialic acid-binding immunoglobulin (Ig)-like lectins (SIGLEC3, SIGLEC14, SIGLEC8), ribosomal proteins (RPL17, RPL34, RPS3A, MRPS33) and chemokines (CCL5, CCL24, CXCL13, CXCL9) were upregulated in Finnsheep, while four multidrug resistance-associated proteins (MRPs) were upregulated in Texel ewes. A total of 17 known genes and two uncharacterized non-coding RNAs (ncRNAs) were differentially expressed in breed-wise comparisons owing to the flushing diet effect. The significantly upregulated TXNL1 gene indicated potential for embryonic diapause in Finnsheep and F1. Moreover, we report, for the first time in any species, several genes that are active in the CL during early pregnancy (including TXNL1, SIGLEC14, SIGLEC8, MRP4, and CA5A).

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