1.Effect of ribosome engineering on butenyl-spinosyns synthesis of Saccharopolyspora pogona.
Lin'gen LUO ; Yan YANG ; Hui WEI ; Jie RANG ; Qiong TANG ; Shengbiao HU ; Yunjun SUN ; Ziquan YU ; Xuezhi DING ; Liqiu XIA
Chinese Journal of Biotechnology 2016;32(2):259-263
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
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Macrolides
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metabolism
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Point Mutation
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Ribosomal Proteins
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genetics
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Ribosomes
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metabolism
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Saccharopolyspora
;
metabolism
2.The connection between tumor and ubiquitin-ribosomal protein S27a, ubiquitin and ribosomal protein.
Chinese Journal of Biotechnology 2007;23(6):982-988
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
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Biomarkers, Tumor
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Cloning, Molecular
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Humans
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Neoplasms
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genetics
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metabolism
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Recombinant Fusion Proteins
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metabolism
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Ribosomal Proteins
;
biosynthesis
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genetics
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Ubiquitin
;
biosynthesis
;
genetics
4.Knockout of ribosomal genes bS22 and bL37 increases the sensitivity of mycobacteria to antibiotics.
Chan SHAN ; Qianwen YUE ; Xiaoming DING
Chinese Journal of Biotechnology 2022;38(3):1061-1073
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*
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Cryoelectron Microscopy
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Mycobacterium/genetics*
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Ribosomal Proteins/genetics*
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Ribosomes/metabolism*
5.Structural insights into the assembly of the 30S ribosomal subunit in vivo: functional role of S5 and location of the 17S rRNA precursor sequence.
Zhixiu YANG ; Qiang GUO ; Simon GOTO ; Yuling CHEN ; Ningning LI ; Kaige YAN ; Yixiao ZHANG ; Akira MUTO ; Haiteng DENG ; Hyouta HIMENO ; Jianlin LEI ; Ning GAO
Protein & Cell 2014;5(5):394-407
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
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Escherichia coli
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metabolism
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Escherichia coli Proteins
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genetics
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metabolism
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GTP Phosphohydrolases
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genetics
;
metabolism
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Mass Spectrometry
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Protein Structure, Secondary
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Protein Structure, Tertiary
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RNA, Ribosomal
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analysis
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metabolism
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Ribosomal Proteins
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chemistry
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genetics
;
metabolism
;
Ribosome Subunits, Small, Bacterial
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chemistry
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metabolism
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ultrastructure
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Salts
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chemistry
6.Localization of polypeptides release factors and ribosome protein L11 in Euplotes octocarinatus.
Baofeng CHAI ; Na LI ; Jingtao WANG ; Quan SHEN ; Zhiyun ZHANG ; Aihua LIANG
Chinese Journal of Biotechnology 2010;26(2):237-243
Protozoan ciliates are a group of unicellular eukaryotes. The special characteristics of stop codons usage in termination of protein biosynthesis in ciliates cells makes them an ideal model to study the mechanism of stop codon recognition of polypeptides release factors. To localize the functional positions of biomolecules in ciliates cell, we constructed a macronuclear artificial chromosome containing a gene encoding red fluorescence protein (EoMAC_R) based on the structural characteristics of ciliates chromosome. Three factors, L11, eRF1a, and eRF3 that are involved in termination process of protein synthesis were colocalized in Euplotes octocarinatus cells by using novel EoMAC_R and the previously constructed EoMAC_G. The results indicated that protein synthesis mainly occurred inside the "C" shape macronucleus, suggesting that EoMAC could be a useful tool for localizing biomolecules in ciliates cell.
Chromosomes, Artificial
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Codon, Terminator
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metabolism
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Euplotes
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chemistry
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Peptide Termination Factors
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analysis
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genetics
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metabolism
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Peptides
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metabolism
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Protein Biosynthesis
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genetics
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Protozoan Proteins
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analysis
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genetics
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Ribosomal Proteins
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analysis
;
genetics
7.Ionizing Radiation-Induced RPL23a Reduction Regulates Apoptosis
Yu Xuan HE ; Yi Xiao GUO ; Yong ZHANG ; Jun Jie HU ; Wei Tao DONG ; Xiang Hong DU ; Xing Xu ZHAO
Biomedical and Environmental Sciences 2021;34(10):789-802
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
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Apoptosis/genetics*
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Gene Expression Regulation
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Male
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Mice
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Ribosomal Proteins/metabolism*
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Signal Transduction
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Spermatogonia/radiation effects*
8.Screening for new binding proteins which interact with BM2 of influenza B virus with yeast two-hybrid system.
Hong YU ; Li-hong YAO ; Ai-jun CHEN ; Jie HE ; Run-qing JIA ; Cong-sheng CHENG ; Zhi-qing ZHANG
Chinese Journal of Experimental and Clinical Virology 2005;19(2):182-184
OBJECTIVETo explore the role of BM2 protein in the life cycle of influenza B virus.
METHODSThe authors screened human kidney MATCHMAKER cDNA library for new binding partners of BM2 of influenza B virus by using the yeast two hybrid system with truncated BM2 (26-109 aa) as the bait.
RESULTSSix positive plasmids encoding N-acetylneuraminate pyruvate lyase, angiopoietin 3, zinc finger protein 251, ribosomal protein S20, protein arginine N-methyltransferase 1 variant 1 (PRMT) and transcription factor-like 1 (TCFL1) were obtained.
CONCLUSIONThe results suggest that BM2 may play an important role in the life cycle of influenza B virus.
Angiopoietin-like Proteins ; Angiopoietins ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Gene Library ; Humans ; Influenza B virus ; genetics ; metabolism ; Kidney ; metabolism ; Oxo-Acid-Lyases ; genetics ; metabolism ; Plasmids ; genetics ; Protein Binding ; Protein-Arginine N-Methyltransferases ; genetics ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Ribosomal Proteins ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Two-Hybrid System Techniques ; Viral Proteins ; genetics ; metabolism ; Zinc Fingers ; genetics
9.Ribosomal protein mutations in Korean patients with Diamond-Blackfan anemia.
Hyojin CHAE ; Joonhong PARK ; Seungok LEE ; Myungshin KIM ; Yonggoo KIM ; Jae Wook LEE ; Nack Gyun CHUNG ; Bin CHO ; Dae Chul JEONG ; Jiyeon KIM ; Jung Rok KIM ; Geon PARK
Experimental & Molecular Medicine 2014;46(3):e88-
Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by hypoproliferative anemia, associated physical malformations and a predisposition to cancer. DBA has been associated with mutations and deletions in the large and small ribosomal protein genes, and genetic aberrations have been detected in approximately50-60% of patients. In this study, nine Korean DBA patients were screened for mutations in eight known DBA genes (RPS19, RPS24, RPS17, RPS10, RPS26, RPL35A, RPL5 and RPL11) using the direct sequencing method. Mutations in RPS19, RPS26 and RPS17 were detected in four, two and one patient, respectively. Among the mutations detected in RPS19, two mutations were novel (c.26T>A, c.357-2A>G). For the mutation-negative cases, array-CGH analysis was performed to identify copy-number variations, and no deletions involving the known DBA gene regions were identified. The relative mRNA expression of RPS19 estimated using real-time quantitative PCR analysis revealed two- to fourfold reductions in RPS19 mRNA expression in three patients with RPS19 mutations, and p53 protein expression analysis by immunohistochemistry showed variable but significant nuclear staining in the DBA patients. In conclusion, heterozygous mutations in the known DBA genes RPS19, RPS26 and RPS17 were detected in seven out of nine Korean DBA patients. Among these patients, RPS19 was the most frequently mutated gene. In addition, decreased RPS19 mRNA expression and p53 overexpression were observed in the Korean DBA patients, which supports the hypothesis that haploinsufficiency and p53 hyperactivation represent a central pathway underlying the pathogenesis of DBA.
Anemia, Diamond-Blackfan/*genetics
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Female
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Gene Frequency
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Humans
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Infant, Newborn
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Male
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*Mutation
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RNA, Messenger/genetics/metabolism
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Republic of Korea
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Ribosomal Proteins/*genetics/metabolism
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Tumor Suppressor Protein p53/genetics/metabolism
10.The effect of pcDNA 3.1/RPs15 on skin fibroblasts in vitro.
Sheng-jian TANG ; Xiao-ke WANG ; Xiao-jie LV ; Xia LIU ; Peng ZHANG ; Jun ZHANG ; Xiao-yan ZHOU
Chinese Journal of Plastic Surgery 2005;21(1):58-61
OBJECTIVETo construct eukaryotic expression vector of ribosomal protein sl5(RPs15) gene and study its effect on mouse skin fibroblasts in vitro.
METHODSThe RPs15 cDNA encoding region of fetal mouse skin was amplified by reverse transcription-polymerase chain reaction (RT-PCR) method and cloned into eukaryotic expression vector pcDNA3.1(-). The recombinant plasmid was transfected into adult mouse skin fibroblasts by FuGENE6 transfection reagents. Then the expression of RPs15 gene, was detected and its biological effect on fibroblasts was measured.
RESULTSThe DNA sequencing result of pcDNA3.1/RPs15 was identical with the reported. The RPs15 gene was expressed in transfected fibroblasts. The growth density of fibroblasts decreased with the conformation changing accordingly.
CONCLUSIONSThe eukaryotic expression vector pcDNA3.1/RPs15 is successively constructed and can be expressed in mouse skin fibroblasts. The results set up a basis for further study of the effect of RPs15 gene on skin fibroblasts.
Animals ; Cells, Cultured ; DNA Repair ; DNA, Complementary ; genetics ; Epithelial Cells ; metabolism ; Fibroblasts ; metabolism ; Genetic Vectors ; Mice ; Nuclear Proteins ; genetics ; Plasmids ; Ribosomal Proteins ; Skin ; cytology ; Transfection