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

Ribosomal engineering is a technique that can improve the biosynthesis of secondary metabolites in the antibiotics-resistant mutants by attacking the bacterial RNA polymerase or ribosome units using the corresponding antibiotics. Ribosomal engineering can be used to discover and increase the production of valuable bioactive secondary metabolites from almost all actinomycetes strains regardless of their genetic accessibility. As a consequence, ribosomal engineering has been widely applied to genome mining and production optimization of secondary metabolites in actinomycetes. To date, more than a dozen of new molecules were discovered and production of approximately 30 secondary metabolites were enhanced using actinomycetes mutant strains generated by ribosomal engineering. This review summarized the mechanism, development, and protocol of ribosomal engineering, highlighting the application of ribosomal engineering in actinomycetes, with the aim to facilitate future development of ribosomal engineering and discovery of actinomycetes secondary metabolites.
Actinobacteria/metabolism* ; Actinomyces/genetics* ; Anti-Bacterial Agents/metabolism* ; Multigene Family ; Ribosomes/genetics*

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*

OBJECTIVETo study the effect of HCV NS5A protein on HCV IRES-dependent translation in HepG2 cells.

METHODSHepG2 cells were co-transfected with a plasmid vector containing a bicistronic transcript carrying Renilla luciferase and firefly luciferase genes separated by HCV IRES sequences, and an expressing vector producing the NS5A protein. The luciferase activity and the mRNA of the luciferase gene were then detected. The NS5A expression was confirmed by fluorescence microscopy.

RESULTSHCV NS5A protein was detected in the cytoplasm of the HepG2 cells transfected with pcDNA-NS5A, and the luciferase activity was up-regulated in the presence of the HCV NS5A protein while the expression of luciferase mRNA showed no difference.

CONCLUSIONHCV NS5A protein can upregulate the HCV IRES activity and this effect is dose-dependent with NS5A.

Hep G2 Cells ; Hepacivirus ; genetics ; metabolism ; Humans ; Plasmids ; Protein Biosynthesis ; Protein Structure, Secondary ; Ribosomes ; metabolism ; Transfection ; Viral Nonstructural Proteins ; metabolism

We used ribosome engineering technology, with which antibiotic-resistant strains are resulted from mutations on microbial ribosome, to screen a high butanol-producing Clostridium strain. A novel mutant strain S3 with high butanol production and tolerance was obtained from the original Clostridium acetobutylicum L7 with the presence of mutagen of streptomycin. Butanol of 12.48 g/L and ethanol of 1.70 g/L were achieved in S3, 11.2% and 50%, respectively higher than the parent strain. The conversion rate of glucose to butanol increased from 0.19 to 0.22, and fermentation time was 9 h shorter. This caused an increase in butanol productivity by 30.5%, reaching 0.24 g/(Lh). The mutant butanol tolerance was increased from 12 g/L to 14 g/L, the viscosity of fermentation broth was dramatically decreased to 4 mPa/s, 60% lower than the parent strain. In addition, the genetic stability of mutant strain S3 was also favorable. These results demonstrate that ribosome engineering technology may be a promising process for developing high butanol-producing strains.
Butanols ; metabolism ; Clostridium acetobutylicum ; drug effects ; genetics ; metabolism ; Fermentation ; Genetic Engineering ; Mutation ; Recombinant Proteins ; biosynthesis ; genetics ; Ribosomes ; genetics ; Streptomycin ; pharmacology

OBJECTIVETo develop a RNAi approach that specifically targets the HCV IRES sequence by vector-expressed short hairpin RNA (shRNA) in vitro, and to assess the inhibitory effect of the shRNA on reporter gene expression.

METHODSEukaryotic expressing plasmids, pIRES-GFP and p5' UTR-Luc containing GFP or luciferase gene controlled by HCV IRES were cotransfected into HepG2 cells with either a RNAi plasmid pshRNA-HCV or a control plasmid pTZU6+1. At 24, 48, 72 hours post transfection, the fluorescence in the transfected cells was studied using fluorescence microscopy. The levels of GFP RNA were determined using RT-PCR and those of protein were determined using Western blot. The activities of luciferase were assayed using a dual luciferase assay system.

RESULTSThe introduction of RNAi plasmid efficiently and specifically down-regulated the expression of the reporter gene. RT-PCR showed that the RNAs of GFP gene were distinctly reduced (about 60%) when the pIRES-GFP was cotransfected with pshRNA-HCV, whereas the control vector did not exhibit inhibitory effect on the mRNA level, according to Western blot assay. The luciferase activity also decreased by 60%-70% in comparison to the control plasmid.

CONCLUSIONOur results demonstrate that the shRNA targeting HCV IRES shows a strong inhibitive effect on the expression of the reporter gene controlled by this sequence, suggesting that RNAi-based anti-HCV strategy may represent a potential approach in the therapy of HCV infection.

Gene Expression Regulation ; Genes, Reporter ; Genetic Therapy ; Genetic Vectors ; Hep G2 Cells ; Hepacivirus ; genetics ; Hepatitis C ; therapy ; Humans ; RNA Interference ; RNA, Messenger ; genetics ; Ribosomes ; genetics ; metabolism ; Transfection

The internal ribosome entry site (IRES) sequence was derived from encephalomyocarditis virus. It allows to translate two open reading frames at one mRNA, so two genes conjoined by IRES have the same expression rate. K(DfGC) and K(DfGd) cell lines, stably expressing D-amino acid oxidase (DAAO) gene and green fluorescence protein (GFP) genes, were obtained by transfection of K562e cells with retroviral vector pLDfG containing IRES sequence, DAAO cDNA and GFP gene. Fluorescence positive rate and fluorescence intensity of the two cell lines were measured with flow cytometry. H(2)O(2) production by K(DfGC) and K(DfGd) cells treated with D-alanine was measured by the phenol red oxidation assay. The fluorescence positive rate and fluorescence intensity in K(DfGC) and K(DfGd) cell were 94.64% and 96.31% and 202 units and 174 units per 2 x 10(4) cells, respectively. There was exponential correlation between fluorescence intensity and H(2)O(2) level. The above-mentioned results demonstrate that DAAO gene and GFP gene were simultaneously expressed in K562e cell line by the regulation of IRES sequence, and DAAO level was correlated with fluorescence intensity of GFP.
Binding Sites ; genetics ; D-Amino-Acid Oxidase ; genetics ; metabolism ; Gene Expression ; Gene Expression Regulation, Enzymologic ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; Humans ; Hydrogen Peroxide ; metabolism ; K562 Cells ; Luminescent Proteins ; genetics ; metabolism ; Retroviridae ; genetics ; Ribosomes ; metabolism ; Transfection

This experimental study was aimed to construct the recombinant bisbicistronic eukaryotic expression vector containing endocrine and exocrine protein (EECP) gene associated with breast cancer and enhanced green fluorescent protein (EGFP) gene. And then we transfected it into breast cancer cells MCF-7 to detect the expression of EECP protein and study preliminary biological function of EECP gene. The EECP sequence was cloned to pBluescript II SK (+) plasmid. After restriction endonuclease reaction of pBluescript II SK(+) plasmid, the EECP fragment was cloned to pIRES2-EGFP vector forming a recombinant eukaryotic expression vector named pEECP-IRES2-EGFP. The potential vector was identified by restriction endonuclease digestion and sequencing. Correct plasmid was extracted and transfected into breast cancer cells MCF-7. The expression of EECP protein was detected by western blot analysis. Its biological function was studied by MTT and Flow-cytometry. It turns out that the recombinant eukaryotic expression vector containing EECP gene and EGFP gene was constructed successfully, and it could transfect MCF-7 cells efficiently. It can get higher expression of EECP protein and higher cell proliferation, thus providing an important and convenient tool for studying the function of EECP gene in vitro and in vivo.
Base Sequence ; Breast Neoplasms ; genetics ; pathology ; Female ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; MCF-7 Cells ; Molecular Sequence Data ; Proteins ; analysis ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Ribosomes ; chemistry ; metabolism

OBJECTIVEIn this study, the authors investigated inhibition of coxsackievirus B (CVB) gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon and structural protein coding sequences and also observed the dose-response of the sequence specific inhibition of CVB plaque formation by antisense oligonucleotides.

METHODSAntiviral activities of these oligonucleotides were evaluated by using plaque reduction assay, yield reduction assay, cytopathic effect (CPE) and Western blot analysis. The cells were treated with random oligonucleotides as a specificity control.

RESULTSAt a screening concentration of 5 micromole, 6 of the phosphorothioate oligonucleotide demonstrated some reduction of virus replication relative to untreated cells. 70%-90% inhibition of virus at 0.1 MOI (multiplicity of infection), 50% inhibition of virus infection at 10 MOI. The levels of the VP1 were reduced in CVB-infected cells treated with Scb561 and Scb733. VP1 was significantly reduced after treatment with 0.625 micromole Scb561 and almost undetectable in cells treated with 2.5 micromole Scb561. Dose response experiments implied that sequence specific oligonucleotide doses were related to effect on inhibition of CVB3 infection. When oligonucleotide doses were increased from 1.25 to 5 micromole, 75% to 90% inhibition were observed with Scb561 and 65% to 80% inhibition with Scb733, whereas random control failed to inhibit CVB replication (8% inhibition for each). CONCLUSION The present studies showed that antisense oligonucleotides against internal ribosome entry site (IRES) and translation initiation codon were capable of specifically inhibiting the synthesis of viral protein and subsequent productive CVB replication.The selective inhibition using antisense oligonucleotide might lead to development of an effective antiviral agent for future clinical evaluation.

5' Untranslated Regions ; genetics ; Dose-Response Relationship, Drug ; Enterovirus B, Human ; drug effects ; genetics ; Gene Expression ; drug effects ; HeLa Cells ; Humans ; Oligonucleotides, Antisense ; pharmacology ; Ribosomes ; metabolism ; Viral Structural Proteins ; biosynthesis ; genetics ; Virus Replication ; drug effects

Adriamycin has been widely used as an anticancer drug in the treatment of thyroid tumor, metastatic breast cancer, sarcoma and lymphoma. The antineoplastic effects of adriamycin result from its inhibitory action on the nucleic acid synthesis and the formation of reactive oxygen radicals by redoxcycling during its metabolic process. Adriamycin affects the normal cells of the patients and causes the undesirable side effects and the toxic effects. Thus, in this study we investigated the effect of dimethyl thiourea (DMTU), a hydroxyl radical scavenger, on the ultrastructural changes of the hepatocyte after the administration of adriamycin in the growing and adult rats. 36 Healthy male Sprague -Dawley adult rats (about 350 g) and 36 male rats at growing peroid (about 120 g) was used as experimental animals. Adriamycin (25 mg/kg) was administered intraperitoneally and DMTU (500 mg/kg) was administered intraperitoneally 30 minutes after the administration of adriamycin. The rats were sacrificed at 24 hours and 72 hours after the administration of adriamycin. A part of the liver from left anterior lobe was obtained and sliced into about 1 mm 3 . The specimens were prepared by the routine methods for electron microscopy. All preparations were stained with uranyl acetate and lead citrate and observed with Hitach -600 electron microscope. The results were as follows. 1. In the hepatocytes of the adult rat dilated, segmented and ribosome detached cisternae of rough endoplasmic reticulum, increased and dilated cisternae of smooth endoplasmic reticulum, damaged mitochondria and autophagic vacuoles were seen after the administration of adriamycin. The ultrastructural changes were progressive with the lapse of time. 2. In the hepatocyte of the growing rat dilated and ribosome detached cisternae of rough endoplasmic reticulum, changes of mitochondria, many lysosomes and the autophagic vacuoles were observed after the administration of adriamycin. 3. DMTU alone did not affect the ultrastructures of the hepatocytes in both growing and adult rats. 4. In the hepatocytes of growing and adult rats, dilated and ribosome detached cisternae of rough endoplasmic reticulum, dilated and increased cisternae of smooth endoplasmic reticulum, changes of mitochondria and autophagic vacuoles were seen after the combined administration of adriamycin and DMTU, but the degree of ultrastructural changes was
Adult* ; Animals ; Breast Neoplasms ; Citric Acid ; Doxorubicin* ; Endoplasmic Reticulum, Rough ; Endoplasmic Reticulum, Smooth ; Hepatocytes* ; Humans ; Hydroxyl Radical ; Liver ; Lymphoma ; Lysosomes ; Male ; Metabolism ; Microscopy, Electron ; Mitochondria ; Rats* ; Reactive Oxygen Species ; Ribosomes ; Sarcoma ; Thiourea ; Thyroid Gland ; Vacuoles