The nucleotide sequence of a region upstream of the type II polyketide synthase genes in the cluster for biosynthesis of the polyketide antibiotic jadomycin B in Streptomyces venezuelae contained an open reading frame encoding a sequence of 196 amino acids that resembeled sequences deduced for a group of repressor proteins. The strongest similarity was to EnvR of Escherichia coli, but the sequence also resembled MtrR, AcrR, TetC, and TcmR, all of which are involved in regulating resistance to antibiotics or toxic hydrophobic substances in the environment. Disruption of the nucleotide sequence of this putative S. venezuelae repressor gene (jadR2), by insertion of an apramycin resistance gene at an internal MluI site, and replacement of the chromosomal gene generated mutants that produced jadomycin B without the stress treatments (exposure to heat shock or to toxic concentrations of ethanol) required for jadomycin B production by the wild type. When cultures of the disruption mutants were ethanol stressed, they overproduced the antibiotic. From these results it was concluded that expression of the jadomycin B biosynthesis genes are negatively regulated by jadR2.
Abstract Global profile of gene expression at single-cell resolution remains to be determined for primates. Using a recently developed technology (“Stereo-seq”), we have obtained a comprehensive single-cell spatial transcriptome map at the whole-brain level for cynomolgus monkeys, with ∼600 genes per cell for 10 μm-thick coronal sections (up to 15 cm 2 in size). Large-scale single-nucleus RNA-seq analysis for ∼1 million cells helped to identify cell types corresponding to Stereo-seq gene expression profiles, providing a 3-D cell type atlas of the monkey brain. Quantitative analysis of Stereo-seq data revealed molecular fingerprints that mark distinct neocortical layers and subregions, as well as domains within subcortical structures including hippocampus, thalamus, striatum, cerebellum, hypothalamus and claustrum. Striking whole-brain topography and coordinated patterns were found in the expression of genes encoding receptors and transporters for neurotransmitters and neuromodulators. These results pave the way for cellular and molecular understanding of organizing principles of the primate brain.
UNLABELLED OBJECTIVE To construct the recombinant plasmid pCI-HLE encoding human serum album-EPO (HSA-EPO) fusion protein and to express it in CHO cell. METHODS The cDNA encoding human serum album and EPO were amplified by PCR, and then spliced with the synsitic DNA fragment encoding GS (GGGGS), by overlap PCR extension to form LEPO. After BamH I digestion, the HSA and LEPO was ligated to generate the fusion HSA-EPO gene and was then cloned into the expression vector pCI-neo to generate the recombinant plasmid pCI-HLE. The plasmid pCI-HLE was transfected into CHO cell by liposome protocol. Then, the recombinant cells were screened by G418 and identified by PCR and Western blot. Expression of fusion protein was evaluated by Enzyme Linked Immunosorbent Assay (ELISA). RESULTS Restrictive enzymes digestion and DNA sequencing revealed that HSA-EPO fusion gene was cloned into expression vector pCI-neo successfully. PCR and Western blot analysis confirmed that the fusion gene was integrated in the genome of CHO cells and expressed successfully. The HSA-EPO production varied from 86 Iu/(mL x 10(6) x 72 h) to 637 IU/(mLx 10(6) x 72 h). CONCLUSION The results confirmed that HSA-EPO fusion gene can be expressed in the CHO cells, with EPO immunogenicity, which could serve as foundation for the development of long-lasting recombinant HSA-EPO protein.
Our previous study showed that the ribosomal protein L21 (RPL21) may play an important role in the development and survival of pancreatic cancer. In this article, RNA interference (RNAi) experiments were performed with RPL21-specific small interfering RNA (siRNA) to elucidate the mechanism by which RPL21 controls PC PANC-1 and BxPC-3 cell proliferation.In the present study, PANC-1, BxPC-3 cells, and BALB/c nude mice were used to investigate antitumor effect and mechanism by which RPL21 controls cell proliferation and apoptosis in vitro and in vivo. The effects of RPL21 knockdown on PANC-1 and BxPC-3 cell proliferation, cell cycle and cell apoptosis in vitro were determined using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays and flow cytometry assay. The mechanism of RPL21 regulating cell proliferation was investigated using transcriptome sequencing analysis and luciferase reporter assay. The effects of RPL21 knockdown on PANC-1 and BxPC-3 cell proliferation in vivo were determined using BALB/c nude mice tumor model.In PANC-1 and BxPC-3 cells, the knockdown of RPL21 expression with corresponding siRNA suppressed cell proliferation in vitro and in vivo, inhibited DNA replication, and induced arrests in the G1 phase of the cell cycle. Further results showed that the mini-chromosome maintenance (MCM) protein family (MCM2-7), CCND1 and CCNE1 were down-regulated significantly in PANC-1 and BxPC-3 cells after transfected with RPL21 siRNA, which suggests that the suppression of DNA replication is due to the reduced expression of MCM2-7 family, and the induction of G1 arrest is correlated with the inhibition of CCND1 and CCNE1. Luciferase reporter assay showed that RPL21 controls the DNA replication and G1-S phase progression possibly through the regulation of E2F1 transcription factor in PC cells. Moreover, RPL21 siRNA showed an apoptosis-inducing effect only in BxPC-3 and PANC-1 cells but not in normal HPDE6-C7 cells. The increase of caspase-8 activities and the loss of mitochondrial membrane potential after RPL21 silencing indicates that the RPL21 gene may be involved in caspase-8-related mitochondrial apoptosis.Our findings suggest that siRNA against the RPL21 gene possesses a potential anti-cancer activity for PC cells by inhibiting their proliferation and DNA replication, as well as inducing cell cycle G1 arrest and cell apoptosis.
A DNA fragment originally detected in Streptomyces venezuelae ISP5230 DNA by hybridization with the actI gene for actinorhodin biosynthesis in Streptomyces coelicolor A3(2), and cloned from the S. venezuelae genome as a 1.8-kb SacI-BglII fragment, was used to probe a Sau3A1 genomic library of S. venezuelae in a lambda vector. Three hybridizing lambda clones contained DNA inserts with overlapping regions. Regions hybridizing with both actI and actIII (a second gene for actinorhodin biosynthesis) were located in the genomic DNA, and were adjacent. Subcloning and sequencing of a 4.9-kb segment of the insert containing these regions identified five open reading frames (ORFs). The deduced products of the ORFs closely resembled in sequence the components of streptomycete type II polyketide synthases (PKSs). From sequence comparisons it was concluded that the ORF1 product encodes a ketoacyl synthase, ORF2 a closely related product probably involved in determining chain length; ORF3 an acyl carrier protein, ORF4 a bifunctional cyclase/dehydrase, and ORF5 a ketoreductase. Integration into the S. venezuelae chromosome of pJV63, containing an insert from the ORF2-ORF4 region, severely depressed jadomycin B biosynthesis. Since the two integrants showed no change in growth or spore pigmentation, the cloned PKS genes are presumed to encode enzymes in the pathway for jadomycin B biosynthesis.
Objective To investigate the effects of multidrug resistance-associated protein(MRP) antisense RNA mediated by recombinant adenoviruses on reversal of the multidrug resistance(MDR) in human drug-resistant hepatocellular carcinoma(HCC) cells.Methods HCC cells of the line SMMC-7721/ADM were cultured in a 96-well-plate and transfected with recombinant adenovirus Ad-ASmrp containing antisense MRP.Another SMMC-7721/ADM cells were transfected with Ad-GFP or added with PBS were used as controls.The cells were collected from 3 wells everyday for 7 days to observe the absorbance so as to draw a growth curve.SMMC-7721/ADM cells were transfected with Ad-ASmrp and Ad-GFP,added with adriamycin(ADM) or daunorubicin(DNR),and then cultured for 48 hours.The chemosensitivity of the cells to ADM and DNR was determined by MTT assay.The levels of MRP mRNA and its product P190 were measured by RT-PCR and flow cytometry(FCM) 24,48,72,96,and 120 hours after respectively.The accumulation of DNR in these cells was analyzed by FCM simultaneously.Results The IC50 values of the cells transfected with Ad-Asmrp to ADM and DNR were 0.487 μg/ml and 0.328μg/ml,and the factors of resistance(RF) decreased by 36.8 and 35.4 respectively(both P0.05).Continuous decrease in levels of MRP mRNA and P190 was observed 24 h and 48h after transfection respectively(both P0.01).The intracellular DNR accumulation was increased simultaneously in the Ad-Asmrp transfected cells(P0.01).Conclusions MRP antisense RNA mediated by recombinant adenovirus increases chemosensitivity and partially reverses the MDR in HCC cells in vitro by inhibiting the expression of MRP gene.
To conduct an in vitro study on the effect of recombinant adenovirus microsphere encapusulated antisense MRP (as-mrp) for use in the gene therapy to overcome drug resistance in hepatocellular carcinoma.Recombinant adenovirus microsphere encapusulated as-mrp was transfected into hepatocellular carcinoma multidrug resistance cells HepG2/ADM, the fluorescence intensity of transfected cells were observed at 48 hours and 120 hours after transfection. in vitro drug sensitivity was measured by MTT assay; the resistant index of andromycin resistant variants was determined by drawing the cell dosage reaction curves. The levels of MRP mRNA expression were detected by RT-PCR and the ratio of MRP mRNA/beta-actin was detected. Intracelluar rubidomycin (DNR) concertration was examined by flow cytometry (FCM).More than 90% of the HepG2/ADM cells could be transfected when microspheres being 10 mg. Adv microsphere inhibited the expression of mRNA in HepG2/ADM and enhanced the sensitivity of HepG2/ADM to chemotherapeutic drug.Recombinant adenovirus microsphere encapusulated as-mrp could effectively reverse HepG2/ADM cells, which would provide an experimental basis for the methods of reversing the multidrug resistance in human hepatocellular carcinoma.
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