Cryoelectron Microscopy ; Mycobacterium smegmatis ; genetics ; ultrastructure ; RNA, Ribosomal ; ultrastructure ; Ribosomal Proteins ; genetics ; isolation & purification ; Ribosomes ; genetics ; ultrastructure

OBJECTIVEThis study aimed to detect the difference in the expression levels of autolysin atIS gene of Streptococcus gordonii (S. gordonii) at different growth stages and pH values, as well as to analyze the factors regulating atlS gene expression in S. gordonii.

METHODSS. gordonii wild strains (ATCC 35105) were collected at different growth stages (early exponential phase, mid-exponential phase, late exponential stage, and platform stage) and pH values (pH 7 and pH 5.5), and total RNA was extracted by using a conventional method. Fluorescence quantitative polymerase chain reaction (FQ-PCR) was used to measure the relative mRNA expression of atlS gene, with bacterial 16S rRNA as internal reference, for a comparison of the mRNA levels of atlS gene expression in S.gordonii at different growth stages and pH values.

RESULTSFQ-PCR results showed that atlS gene expression increased with gradually increasing growth stage under neutral conditions and was higher than that under acidic conditions (P < 0.05).

CONCLUSIONThe atlS gene expression in S. gordonii is influenced by growth stage and pH value factors.

OBJECTIVETo investigate the clinical features of Diamond-Blackfan anemia (DBA) and related pathogenic genes.

METHODSA retrospective analysis was performed for the clinical data of two children with DBA, and related literature was reviewed.

RESULTSThe two children with DBA (2-3 months old) manifested with severe normochromic normocytic anemia, decreased reticulocyte count, and increased serum iron and serum ferritin. Normal white blood cell and platelet counts were noted in the two patients. Bone marrow examination showed a decreased percentage of erythrocytes and rare normoblasts in the two patients. Gene screening showed a reported pathogenic heterozygous mutation in RPS19 gene, c.212G>A (p. Gly71Glu), in one patient, and there were no mutations in his parents. In the other patient, gene screening showed a heterozygous mutation in RPL5 gene, c.740T>C (p. I247L), which had not been reported in literature, and there were no mutations in her parents. A bioinformatic analysis showed that this might be a pathogenic mutation.

CONCLUSIONSThe onset age of DBA is early infancy in most children, with a manifestation of erythroid deficiency. RPS19 and RPL5 gene mutations are common causes of this disease. Molecular detection helps with the early diagnosis of DBA.

Through introducing mutations into ribosomes by obtaining spontaneous drug resistance of microorganisms, ribosome engineering technology is an effective approach to develop mutant strains that overproduce secondary metabolites. In this study, ribosome engineering was used to improve the yield of butenyl-spinosyns produced by Saccharopolyspora pogona by screening streptomycin resistant mutants. The yields of butenyl-spinosyns were then analyzed and compared with the parent strain. Among the mutants, S13 displayed the greatest increase in the yield of butenyl-spinosyns, which was 1.79 fold higher than that in the parent strain. Further analysis of the metabolite profile of S13 by mass spectrometry lead to the discovery of Spinosyn α1, which was absent from the parent strain. DNA sequencing showed that there existed two point mutations in the conserved regions of rpsL gene which encodes ribosomal protein S12 in S13. The mutations occurred a C to A and a C to T transversion mutations occurred at nucleotide pair 314 and 320 respectively, which resulted in the mutations of Proline (105) to Gultamine and Alanine (107) to Valine. It also demonstrated that S13 exhibited genetic stability even after five passages.
Genetic Engineering ; Macrolides ; metabolism ; Point Mutation ; Ribosomal Proteins ; genetics ; Ribosomes ; metabolism ; Saccharopolyspora ; metabolism

In recent years, two novel proteins in the ribosomes of mycobacteria have been discovered by cryo-electron microscopy. The protein bS22 is located near the decoding center of the 30S subunit, and the protein bL37 is located near the peptidyl transferase center of the 50S subunit. Since these two proteins bind to conserved regions of the ribosome targeted by antibiotics, it is speculated that they might affect the binding of related drugs to these targets. Therefore, we knocked out the genes encoding these two proteins in wild-type Mycolicibacterium smegmatis mc²155 through homologous recombination, and then determined the growth curves of these mutants and their sensitivity to related antibiotics. The results showed that compared with the wild-type strain, the growth rate of these two mutants did not change significantly. However, mutant ΔbS22 showed increased sensitivity to capreomycin, kanamycin, amikacin, streptomycin, gentamicin, paromomycin, and hygromycin B, while mutant ΔbL37 showed increased sensitivity to linezolid. These changes in antibiotics sensitivity were restored by gene complementation. This study hints at the possibility of using ribosomal proteins bS22 and bL37 as targets for drug design.
Anti-Bacterial Agents/pharmacology* ; Cryoelectron Microscopy ; Mycobacterium/genetics* ; Ribosomal Proteins/genetics* ; Ribosomes/metabolism*

Objective: The expression patterns of ribosomal large subunit protein 23a (RPL23a) in mouse testes and GC-1 cells were analyzed to investigate the potential relationship between RPL23a expression and spermatogonia apoptosis upon exposure to X-ray. Methods: Male mice and GC-1 cells were irradiated with X-ray, terminal dUTP nick end-labelling (TUNEL) was performed to detect apoptotic spermatogonia Results: Ionizing radiation (IR) increased spermatogonia apoptosis, the expression of RPL11, MDM2 and p53, and decreased RPL23a expression in mice spermatogonia Conclusion These results suggested that IR reduced RPL23a expression, leading to weakened the RPL23a-RPL11 interactions, which may have activated p53, resulting in spermatogonia apoptosis. These results provide insights into environmental and clinical risks of radiotherapy following exposure to IR in male fertility. The graphical abstract was available in the web of www.besjournal.com.
Animals ; Apoptosis/genetics* ; Gene Expression Regulation ; Male ; Mice ; Ribosomal Proteins/metabolism* ; Signal Transduction ; Spermatogonia/radiation effects*

Ubiquitin-ribosomal protein S27a(UBRPS27a) is a fusion protein of Ubiquitin and ribosomal protein. The N-terminal is ubiquitin and C-termina is ribosomal protein S27a with a high conservative zinc finger domain of the C2-C2 type. When it was expressed in eukaryotes,The intact fusion protein were rapidly processed to free ubiquitin monomer and ribosomal protein S27a (RPS27a). Ubiquitin degradated proteins particularly and selectively in cell and RPS27a is indispensable for translation. This multifunctional ribosomal protein is expressed at high levels in a wide variety of actively proliferating cells and tumor tissues and is a representative characteristic of various tumor cells. In our preliminary study of this protein in the silkworm,RPS27a also be found express highly in actively proliferating cells. The precise functional role of each ribosomal protein is largely unknown and many ribosomal proteins have extraribosomal functions apart from the particle. In this article, we review the recent research on the connection between tumor and this fusion protein, Ubiquitin-Proteasome Pathway and ribosomal protein. These research may indicate the origin and development of tumor, provide the basis for clinical diagnosis of cancer and the novel therapeutic targets for the treatment of malignant tumors.
Animals ; Biomarkers, Tumor ; Cloning, Molecular ; Humans ; Neoplasms ; genetics ; metabolism ; Recombinant Fusion Proteins ; metabolism ; Ribosomal Proteins ; biosynthesis ; genetics ; Ubiquitin ; biosynthesis ; genetics

PURPOSE: When murine myeloma cells P3-X63-Ag8.653 (V653) of this model treated with amin opterin, an anticancer drug, they can't synthesize nucleic acid via de novo or salvage pathway and selectively eliminated due to apoptosis. This study was aimed to clone specific known and novel genes preferentially expressed in aminopteirn-treated tumor cell apoptosis. MATERIALS AND METHODS: This study was aimed to clone specific known and novel genes pre ferentially expressed in aminopteirn-treated tumor cell apoptosis by using subtraction-PCR technique. RESULTS: By using this technique 868 clones were obtained. Of these 427 clones were positive with insert DNA. By using cross-hybridization Southern blotting, final 101 clones were selected. All of these genes were sequenced and analyzed by using genebank DNA database. Total 101 clones of genes preferentially expressed in apoptotic tumor cells were classified into 10 groups, which included ribosomal proteins, nuclear proteins, mitdegrees Chondrial proteins, signal transductional proteins, retroviral proteins, cell surface receptor proteins, cell structural proteins, unclassified miscellaneous proteins, and novel genes. Especially, Unknown novel genes preferentially ex pressed in this apoptotic tumor cells included clone numbers S1-63, 1-1, 1-3, 1-16, 1-18, 1-20, 3-33, 3-41, 3-44, 3-48, 3-55, 3-60, 6-17, 6-25, 8-12, 50-7, 50-23, and 100-35. CONCLUSION: It seemed that known and unknown novel genes cloned in this study would con tribute to the future studies regarding apoptosis of tumor cells and cancer treatment therepy.
Aminopterin ; Apoptosis* ; Blotting, Southern ; Clone Cells* ; Cloning, Organism* ; Databases, Nucleic Acid ; DNA ; Mass Screening* ; Membrane Proteins ; Nuclear Proteins ; Ribosomal Proteins ; Zidovudine

Low molecular proteins (LMPs) which are smaller than 20 kDa are difficult to visible on a standard two-dimensional SDS-polyacrylamide gel electrophoresis (2-D SDS-PAGE) map. LMPs must be enriched appropriately to be analyzed. We isolated LMPs of Helicobacter pylori 26695 from 1-D polyacrylamide gel and digested by pepsin. Pepsin-digested LMPs were separated by HPLC and each fraction was analyzed by hybrid tandem mass spectrometer. Seventy nine peptides, representing 27 genes, including copper ion binding protein (CopP, 7 kDa), thioredoxin (TrxA, 11.9 kDa) and ribosomal protein L23 (Rpl23, 10.5 kDa) were identified. Some proteins larger than 40 kDa including Omp2, Omp21, Omp27, Omp30, Omp32, catalase and HP1083 were also identified. This work may give researchers a useful way to analyse the expressed LMPs which could not be identified on the conventional 2-D SDS-PAGE.
Acrylic Resins ; Carrier Proteins ; Catalase ; Chimera ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Copper ; Electrophoresis ; Electrophoresis, Polyacrylamide Gel ; Helicobacter pylori ; Molecular Weight ; Pepsin A ; Peptides ; Proteins ; Proteome ; Ribosomal Proteins ; Thioredoxins

The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coli strain (∆rsgA∆rbfA). The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, intermediates derived under the two contrasting salt conditions (high and low) likely reflect two distinctive assembly stages, the relatively early and late stages of the 3' domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5' end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal the location of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mechanisms on subunit production and protein translation.
Cryoelectron Microscopy ; Escherichia coli ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; GTP Phosphohydrolases ; genetics ; metabolism ; Mass Spectrometry ; Protein Structure, Secondary ; Protein Structure, Tertiary ; RNA, Ribosomal ; analysis ; metabolism ; Ribosomal Proteins ; chemistry ; genetics ; metabolism ; Ribosome Subunits, Small, Bacterial ; chemistry ; metabolism ; ultrastructure ; Salts ; chemistry