Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.     

基于CRISPR/Cas9系统构建精准调控Wnt信号通路的表达载体及其效果的验证

目的:构建基于CRISPR/cas9系统调控Wnt信号通路的载体,并在细胞水平验证其调控基因表达的效率。方法:选取Wnt信号中负性调控分子,设计并合成能够表达靶向上述分子gRNA的互补DNA克隆序列,BsmBI限制性内切酶酶切载体后,采用分子克隆的方法将上述序列克隆至目的载体lenti-sgRNA-Ms2-zeo,测序正确的克隆通过Lipofectamine2000与lenti-Ms2-P65-HSF1-Hygro和lenti-dcas9-VP64-blastine共同转染入293细胞;转染24h后收集细胞,qRt-PCR检测目的基因的表达。结果:筛选了Wnt信号通路中已知的19个负性调控基因;针对每个基因设计了两对gRNA序列,并构建了能够表达gRNA和MS2融合序列的载体,测序结果显示重组质粒的DNA序列与预期完全相符。随机挑选了4个表达载体与lenti-Ms2-P65-HSF1-Hygro和lenti-dcas9-VP64-blastine共转进入细胞,qPCR结果显示构建的目的载体联合lenti-Ms2-P65-HSF1-Hygro和lenti-dcas9-VP64-blastine载体可以协同促进靶分子表达。结论:本研究成功构建了基于CRISPR/cas9基因编辑系统调控Wnt信号的载体。     

Gene fusion vectors based on the gene for staphylococcal protein A

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene, thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the lacZ gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and β-galactosidase activities. The hybrid protein(s) can be immobilized on IgG-Sepharose by its protein A moiety with high efficiency without losing its enzymatic activity and they can be eluted from the column by competitive elution with pure protein A. The fused protein(s) also binds to IgG-coated microtiter wells which means that the in vivo product can be used as an enzyme conjugate in ELISA tests.     

Observer design for differential-algebraic model of an aerobic culture of a recombinant yeast

The mathematical model of an aerobic culture of recombinant yeast presented in work by Zhang et al. (1997) is given by a differential-algebraic system. The classical nonlinear observer algorithms are generally based on ordinary differential equations. In this paper, first we extend the nonlinear observer synthesis to differential-algebraic dynamical systems. Next, we apply this observer theory to the mathematical model proposed in Zhang et al. (1997). More precisely, based on the total cell concentration and the recombinant protein concentration, the observer gives the online estimation of the glucose, the ethanol, the plasmid-bearing cell concentration and a parameter that represents the probability of plasmid loss of plasmid-bearing cells. Numerical simulations are given to show the good performances of the designed observer.Symbols C ₁ activity of pacing enzyme pool for glucose fermentation (dimensionless) - C ₂ activity of pacing enzyme pool for glucose oxidation (dimensionless) - C ₃ activity of pacing enzyme pool for ethanol oxidation (dimensionless) - E ethanol concentration (g/l) - G glucose concentration (g/l) - k _a regulation constant for (g glucose/g cell h^–1) - k _b regulation constant for (dimensionless) - k _c regulation constant for (g glucose/g cell h^–1) - k _d regulation constant for (dimensionless) - K _m1 saturation constant for glucose fermentation (g/l) - K _m2 saturation constant for glucose oxidation (g/l) - K _m3 saturation constant for ethanol oxidation (g/l) - L ( t) time lag function (dimensionless) - p probability of plasmid loss of plasmid-bearing cells (dimensionless) - P recombinant protein concentration (mg/g cell) - q _G total glucose flux culture time (g glucose/g cell h) - t culture time (h) - t _lag lag time (h) - X total cell concentration (g/l) - X ⁺ plasmid-bearing cell concentration (g/l) - Y ^F _{X / G} cell yield for glucose fermentation pathway (g cell/g glucose) - Y ^O _{X / G} cell yield for glucose oxidation pathway (g cell/g glucose) - Y _{X / E} cell yield for ethanol oxidation pathway (g cell/g ethanol) - Y _{E / X} ethanol yield for fermentation pathway based on cell mass (g ethanol·g cell) - ₂ glucoamylase yield for glucose oxidation (units/g cell) - ₃ glucoamylase yield for ethanol oxidation (units/g cell) - µ₁ specific growth rate for glucose fermentation (h^–1) - µ₂ specific growth rate for glucose oxidation (h^–1) - µ₃ specific growth rate for ethanol oxidation (h^–1) - µ_1max maximum specific growth rate for glucose fermentation (h^–1) - µ_2max maximum specific growth rate for glucose oxidation (h^–1) - µ_3max maximum specific growth rate for ethanol oxidation (h^–1)     

Purification of restriction endonuclease XcyI from Xanthomonas cyanopsidis

A new Type II restriction endonuclease XcyI, purified from Xanthomonas cyanopsidis 13D5, is an isoschizomer of SmaI and XmaI that cleaves at the nucleotide sequence 5'-C decreases CCGGG-3' of double-stranded DNA. The single restriction activity present in this strain permits rapid purification of 8000 units of cleavage activity from 10 g of freshly harvested cells. The resulting XcyI preparation is free of contaminating nuclease activities that interfere with in vitro manipulation of DNA.     

Replication and packaging of Turnip yellow mosaic virus RNA containing Flock house virus RNA1 sequence

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. In this study, RNA1 sequence of Flock house virus (FHV) was inserted into the TYMV genome to test whether TYMV can accommodate and express another viral entity. In the resulting construct, designated TY-FHV, the FHV RNA1 sequence was expressed as a TYMV subgenomic RNA. Northern analysis of the Nicotiana benthamiana leaves agroinfiltrated with the TY-FHV showed that both genomic and subgenomic FHV RNAs were abundantly produced. This indicates that the FHV RNA1 sequence was correctly expressed and translated to produce a functional FHV replicase. Although these FHV RNAs were not encapsidated, the FHV RNA having a TYMV CP sequence at the 3’-end was efficiently encapsidated. When an eGFP gene was inserted into the B2 ORF of the FHV sequence, a fusion protein of B2-eGFP was produced as expected. [BMB Reports 2014; 47(6): 330-335]     

Ligand-receptor interactions as controlled by wild-type and mutant Thr(370)Lys alpha2B-adrenoceptor-Galpha15 fusion proteins

Fusion proteins were constructed between either a wild-type or mutant Thr370Lys alpha2B-adrenoceptor (alpha2B AR) and a mouse Galpha15 protein to analyze ligand-receptor interactions at a receptor/Galpha15 protein density ratio of 1. Activation of the wild-type alpha2B AR-Galpha15 fusion protein in CHO-K1 cells by (-)-adrenaline induced a time- and concentration-dependent (pEC50 = 7.37+/-0.13) increase in the intracellular Ca2+ concentration, which could be antagonized by RX 811059 (pK(B) = 7.55+/-0.15). Whereas d-medetomidine and oxymetazoline were as efficacious agonists as (-)-adrenaline, the following ligands displayed partial agonist properties: BRL 44408 < atipamezole < clonidine < UK 14304 < BHT 920. A comparison with the mutant Thr370Lys alpha2B AR-Galpha15 fusion protein displayed similar Ca2+ kinetics and a ligand-mediated receptor activation profile characterized by higher potencies and greater maximal Ca2+ responses for the ligands being investigated, including the putative antagonists dexefaroxan and idazoxan. RX 811059 and RX 821002 remained silent. Similar conclusions could be made on enhancement of the ligands' intrinsic activities by coexpression of the mutant Thr370Lys alpha2B AR with either a Galpha15 or Galphao Cys351Ile protein. The Thr370Lys alpha2B AR-Galpha protein interactions may modify the tertiary structure of the mutant receptor in such a way that some putative alpha2 AR antagonists are capable of stabilizing an active receptor conformation, thereby generating positive efficacy.     

Whole-cell biocatalysis: Evaluation of new hydrophobic ionic liquids for efficient asymmetric reduction of prochiral ketones

A biphasic process design is often applied in whole-cell biocatalysis if substrate and product have low water solubility, are unstable in water or toxic for the biocatalyst. Some water immiscible ionic liquids (ILs) with adequate distribution coefficients have already been applied successfully as second liquid phase, which acts as a substrate reservoir and in situ extractant for the product. In this work, 12 new ILs were evaluated with respect to their applicability in biphasic asymmetric reductions of prochiral ketones in comparison to 9 already published ILs. The ILs under study are composed of seven different cations and three different anions. Recombinant Escherichia coli was used as whole-cell biocatalyst overexpressing the genes of a Lactobacillus brevis alcohol dehydrogenase (LB-ADH) and a Candida boidinii formate dehydrogenase (CB-FDH) for cofactor regeneration. Best results were achieved if ionic liquids with [PF6]- and [NTF]-anions were applied, whereas [FAP]-ILs showed minor qualification, e.g., the use of [HMPL][NTF] as second liquid phase for asymmetric synthesis of (R)-2-octanol resulted in a space–time-yield of 180 g L⁻¹ d⁻¹, a chemical yield of 95% and an enantiomeric excess of 99.7% in a simple batch process.