Spontaneous mutations in the galactose operons of Streptomyces coelicolor A3 (2) and Streptomyces lividans 66

Mutations can be divided into two classes: point mutations (base changes, frame shifts) and DNA rearrangements (deletions, insertions, inversions etc.). In Escherichia coli K 12, DNA insertions account for up to 40% of spontaneous mutations that inactivate genes (for review, see CULLUM 1985) and the Insertion Sequences involved can also mediate deletions and inversions. We started to study spontaneous mutations in Streptomyces in the expectation of isolating transposable elements and chose the galactose genes as convenient system. When selection is made for resistance to the galactose analogue 2-deoxygalactose (2dgalR), gal K mutants are obtained that are defective in the enzyme galactokinase (Kendall et al. 1987). We isolated spontaneous 2dgalR mutations in the closely related strain S. coelicolor A 3 (2) and S. lividans 66 and cloned the gal K gene of the former strain. Adams et al. (1988) have cloned and sequenced the gal genes of S. lividans 66 and shown that they form an operon, the restriction map appears to be identical to that of S. coelicolor A 3 (2). In this paper we describe the characterisation of spontaneous 2 dgalR mutants in S. coelicolor A 3 (2) and S. lividans 66.     

Review of the Streptomyces lividans/vector pIJ702 system for gene cloning.

Interest in the biology of the Streptomyces and application of these soil bacteria to production of commercial antibiotics and enzymes has stimulated the development of efficient cloning techniques and a variety of streptomycete plasmid and phage vectors. Streptomyces lividans is routinely employed as a host for gene cloning, largely because this species recognizes a large number of promoters and appears to lack a restriction system. Vector pIJ702 was constructed from a variant of a larger autonomous plasmid and is often used as a cloning vehicle in conjunction with S. lividans. The host range of vector pIJ702 extends beyond Streptomyces spp., and its high copy number has been exploited for the overproduction of cloned gene products. This combination of host and vector has been used successfully to investigate antibiotic biosynthesis, gene structure and expression, and to map various Streptomyces mutants.     

Distribution functions of variables characterizing the mycelial morphology of Streptomyces hygtroscopicus grown in glucose-limited chemostat cultures

The distribution of variables characterizing the morphology of the mycelium of Streptomyces hygroscopicus grown in glucose-limited chemostat cultures at different specific growth rates were investigated statistically. The values of the hyphal growth unit (L/N) and the values of the distance from the apex to the first branch (L_p) are normally distributed, but the values of the distance between neighbouring branches are logarithmically normal distributed. The distribution functions are discussed from the biological point of view.     

重组质粒pZeoSV2(+)/CpG-HBcAg(ISS)对Balb/c小鼠的免疫作用

目的探讨重组质粒pZeoSV2(+)/CpG-HBcAg(ISS)对Balb/c小鼠免疫的作用。方法构建真核表达质粒pZeoSV2(+)/CpG-HBcAg(ISSb,ISSc),将其免疫Balb/c小鼠,ELISA法检测免疫后小鼠血清中HBcAb、IFN-γ、IL-2、IL-12、IL-4和IL-10的含量。结果 pZeoSV2(+)/CpG-HBcAg(ISSb,ISSc)重组质粒均能诱导Balb/c小鼠产生特异性抗体HBcAb,pZeoSV2(+)/CpG-HBcAg(ISSb)组较pZeoSV2(+)/CpG-HBcAg(ISSc)组产生的抗-HBc效价显著增高(P<0.01),且其免疫Balb/c小鼠后能使Th1型细胞因子IFN-γ、IL-2和IL-12的表达增强,抑制Th2型细胞因子IL-4和IL-10的产生。结论 pZeoSV2(+)/CpG-HBcAg(ISSb,ISSc)重组质粒对小鼠HBcAb的产生具有明显的促进作用,含CpG-HBcAg(ISSb)的重组质粒能够明显增强小鼠血清Th1型细胞因子的表达,抑制Th2型细胞因子的表达。     

Review of the Streptomyces lividans/Vector plJ702 System for Gene Cloning

Abstract

Interest in the biology of the Streptomyces and application of these soil bacteria to production of commercial antibiotics and enzymes has stimulated the development of efficient cloning techniques and a variety of streptomycete plasmid and phage vectors. Streptomyces lividans is routinely employed as a host for gene cloning, largely because this species recognizes a large number of promoters and appears to lack a restriction system. Vector pIJ702 was constructed from a variant of a larger autonomous plasmid and is often used as a cloning vehicle in conjunction with S. lividans. The host range of vector pIJ702 extends beyond Streptomyces spp., and its high copy number has been exploited for the overproduction of cloned gene products. This combination of host and vector has been used successfully to investigate antibiotic biosynthesis, gene structure and expression, and to map various Streptomyces mutants.     

Stability of Streptomyces hygrosocpicus IMET JA 6599 under different cultivation conditions in the chemostat

The stability of the macrolide antibiotic-producing strain Streptomyces hygroscopicus IMET 6599 (wild type) was in vestigated during cultivation in a chemostat with glucose limitation. No changes in the antibiotic-producing ability were found. But under special experimental conditions which consist of a sequence of nearly steady states interrupted by batch processes linked with excess of glucose, colonies with vigorous changed morphology and a green pigment after transfer to surface culture were obtained. The antibiotic activity of the wild type and of the green selectant decreased during the special experimental conditions. The formation of the green selectant is discussed in connection with changes in the glucose or energy metabolism.     

人PLAGL2基因表达载体质4粒的构建

目的克隆人PLAGL2基因片段并插入（TetO）7-CMV表达载体。方法采用PCR技术扩增出人PLAGL2基因的两个DNA片段，经酶连后获得约2kb的目的DNA片段并插入（TetO）7-CMV载体中，PCR筛选阳性转化株并分析重组质粒的DNA序列。结果DNA测序结果显示重组质粒（TetO）7-CMV-PLAGL2序列正确。结论扩增并连接了人PLAGL2基因的两个DNA片段，最终成功构建了人PLAGL2基因的表达载体质粒-（TetO）7-CMV-PLAGL2。     

Plasmid-dependent co-mutation in Streptomyces coelicolor A3(2)

Summary A spontaneous chromosomalmutation(plc A^–) in Streptomyces coelicolor A3(2) made the inborn plasmid SCP1 susceptible to curing by UV irradiation. Lack of the SCP1 plasmid (in SCP1^– strains) prevented the occurrence of the co-mutation process after MNNG treatment, although it left the susceptibility to the lethal effect of the mutagen virtually unaffected. SCP1^– strains were, however, ultrasensitive to the lethal effect of UV. Curing a plc A^– strain of its SCPI plasmid made it refractory to co-mutation by MNNG and sensitive to the lethal effect of UV; reinfected by the plasmid, the strain resumed both the co-mutation proficiency and the UV-resistance. The occurrence on the SCP1 plasmid of a gene comparable to the uvrE gene of E. coli (Nevers and Spatz 1975) was assumed.     

Role of the Streptomyces spore wall synthesizing complex SSSC in differentiation of Streptomyces coelicolor A3(2)

A crucial stage of the Streptomyces life cycle is the sporulation septation, a process were dozens of cross walls are synchronously formed in the aerial hyphae in a highly coordinated manner. This process includes the remodeling of the spore envelopes to make Streptomyces spores resistant to detrimental environmental conditions. Sporulation septation and the synthesis of the thickened spore envelope in S. coelicolor A3(2) involves the Streptomyces spore wall synthesizing complex SSSC. The SSSC is a multi-protein complex including proteins directing peptidoglycan synthesis (MreBCD, PBP2, Sfr, RodZ) and cell wall glycopolymer synthesis (PdtA). It also includes two eukaryotic like serin/threonine protein kinases (eSTPK), PkaI and PkaH, which were shown to phosphorylate MreC. Since unbalancing phosphorylation activity by either deleting eSTPK genes or by expressing a second copy of an eSTPK gene affected proper sporulation, a model was developed, in which the activity of the SSSC is controlled by protein phosphorylation.     

Interferon (IFN) therapy (recombinant IFN-alpha-2C or recombinant IFN-gamma) in metastasized hypernephroma

Recombinant interferon-alpha-2C (rec. IFN alpha-2C) and recombinant interferon-gamma (IFN-gamma) was studied in 12 patients with metastasized renal cell carcinoma. 8 patients were treated with IFN-alpha-2C: 1 patient achieved a complete remission, 2 patients showed mixed responses, and 2 had stabilisation of their disease. In 3 patients progressive disease was observed. IFN-gamma was studied in 4 patients; 2 patients showed stable and 2 progressive disease. Side effects of IFN-alpha treatment included influenza-like symptoms, moderate hematological toxicity and neurological symptoms. During treatment with IFN-gamma similar side effects were observed, although fever generally was more pronounced. All symptoms ceased after dosis reduction or discontinuation of treatment.     

Induction of phenol assimilation in chemostat cultures of Candida maltosa L4

To investigate the induction of phenol hydroxylase and catechol 1,2-dioxygenase in Candida maltosa L4 during chemostat cultivation, changes in the growth-limiting substrate were carried out at different dilution rates. No activity of phenol hydroxylase and catechol 1,2-dioxygenase could be detected in the cells during chemostat cultivation of C. maltosa L4 under glucose limitation independent of the dilution rate. However, once the feed line was changed from glucose medium (s_r = 28 mM) to a medium containing either phenol (s_r = 28 mM) or a mixture of phenol (s_r = 18 mM) and glucose (s_r = 10 mM), both the phenol hydroxylase and the catechol 1,2-dioxygenase were fully induced. After 5 hours at all dilution rates tested (D = 0.05 h⁻¹, D = 0.11 h⁻¹, and D = 0.2 h⁻¹). In spite of this enzyme synthesis, the cells were washed out if C. maltosa L4 was shifted from glucose to phenol at a higher dilution rate than D = 0.05 h⁻¹, probably due to accumulation of inhibiting concentrations of phenol in the medium and starvation of NADPH. If being shifted from glucose to a mixture of phenol and glucose, however, the cells reached a new steady state even at a higher dilution rate of D = 0.2 h⁻¹. The specific activity of phenol hydroxylase in crude extracts of C. maltosa L4 was nearly independent of the growth rate in the range D = 0.05 h⁻¹ to D = 0.2 h⁻¹ on both phenol and a mixture of phenol and glucose, respectively, as the growth-limiting substrates. In contrast, catechol 1,2-dioxygenase activity correlated with the growth rate when the cells were cultivated on phenol. In the presence of mixtures of phenol and glucose, the observed decrease of specific activity of this enzyme at a high dilution rate (D = 0.2 h⁻¹) is possibly a result of repression by glucose or an glucose metabolite.     

HPV18E2蛋白CTL表位多肽的构建与表达

目的分析HPV18E2蛋白的CTL表位,合成并纯化这些表位多肽,构建表达HPV18E2蛋白的靶细胞,检验特异性CTL对靶细胞的杀伤效果,为进一步研制人乳头瘤病毒18型（HPV18）基因工程疫苗打下基础。方法以重组质粒（pBR322-HPV18）为模板,利用PCR方法扩增HPV18E2 DNA片段,将HPV18E2 DNA与加强绿色荧光质粒（pIRES2-EGFP）重组构建重组质粒（pIRES2-HPV18E2-EGFP）。用酶切电泳及测序检查质粒重组后序列正确性。重组质粒转染Hela细胞。51^Cr释放实验评估CTL杀伤能力。结果克隆重组质粒pIRES2-HPV18E2-EGFP酶切后显示的酶切图谱与预期相同,而且测序验证插入片段全序列无改变。转染并用G418筛选后,在荧光显微镜下可见绿色荧光细胞的表达。三条候选多肽特异性杀伤能力分别为60.00%、71.29%和80.00%。结论三条备选肽段都具有明显的杀伤效应,均为HPV18E2蛋白的有效CTL表位。     

柯萨奇B4病毒非结构蛋白P2C重组质粒的构建

目的：构建柯萨奇B4病毒非结构蛋白P2C重组质粒。方法：用RT-PCR技术，从柯萨奇B4病毒中钓取非结构蛋白P2C cDNA片段，克隆入PUCan-T载体，转化大肠杆菌DH5α，构建重组质粒。结果：以柯萨奇B4病毒的总RNA为模板，扩增出987bp大小的片段，克隆入PUCan-T载体，用BamHⅠ、HindⅢ双酶切鉴定，与非结构蛋白P2C的PCR扩增产物片段大小一致。结论：成功地构建了柯萨奇B4病毒非结构蛋白P2C重组质粒。     

Genome-scale analysis of Streptomyces coelicolor A3(2) metabolism

Streptomyces are filamentous soil bacteria that produce more than half of the known microbial antibiotics. We present the first genome-scale metabolic model of a representative of this group--Streptomyces coelicolor A3(2). The metabolism reconstruction was based on annotated genes, physiological and biochemical information. The stoichiometric model includes 819 biochemical conversions and 152 transport reactions, accounting for a total of 971 reactions. Of the reactions in the network, 700 are unique, while the rest are iso-reactions. The network comprises 500 metabolites. A total of 711 open reading frames (ORFs) were included in the model, which corresponds to 13% of the ORFs with assigned function in the S. coelicolor A3(2) genome. In a comparative analysis with the Streptomyces avermitilis genome, we showed that the metabolic genes are highly conserved between these species and therefore the model is suitable for use with other Streptomycetes. Flux balance analysis was applied for studies of the reconstructed metabolic network and to assess its metabolic capabilities for growth and polyketides production. The model predictions of wild-type and mutants' growth on different carbon and nitrogen sources agreed with the experimental data in most cases. We estimated the impact of each reaction knockout on the growth of the in silico strain on 62 carbon sources and two nitrogen sources, thereby identifying the "core" of the essential reactions. We also illustrated how reconstruction of a metabolic network at the genome level can be used to fill gaps in genome annotation.     

A recombinant plasmid carrying the mitochondrial plasmid sequence of Neurospora intermedia LaBelle yields new plasmid derivatives in Neurospora crassa transformants

Summary We have studied the efficacy of transformation of Neurospora crassa with a chimaeric plasmid. We constructed a recombinant plasmid, pMK2, consisting of the mitochondrial plasmid of N. intermedia LaBelle, a part of the qa gene cluster of Neurospora and the Escherichia coli plasmid pBR322. Compared to plasmid pVK88, not containing the LaBelle sequence, the pMK2 plasmid gives a five-fold increase in transformation of the qa2⁺ gene. Analysis of the DNA from Neurospora transformants revealed that the pMK2 plasmid is not stable. The qa insert as well as the LaBelle part of pMK2 are rapidly lost from the plasmid. In most cases the qa insert integrates into the nuclear DNA of the host. Plasmids recovered from Neurospora transformants are rearranged and show insertions or deletions. Some of these plasmids are described here. In most cases the qa insert and the LaBelle sequence of plasmid pMK2 have been deleted. Frequently plasmid dimers, carrying an insertion of mitochondrial DNA, are recovered.     

Bistability in the glucose and energy metabolism of ammonia-limited chemostat cultures of Escherichia coli ML 30

Recently bistability in the pyruvate production of ammonia-limited glucose-grown Escherichia coli ML 30 chemostat cultures was evidenced. The present investigation shows that the state of higher pyruvate production is connected with a lower glycogen content than in cells in the state of lower pyruvate production. The results support the view of a regulation of bistability on the step of pyruvate consumption by the citric acid cycle. Pronounced differences in energy formation in the two states of glucose bistability are discussed.     

The pharmacokinetics of recombinant alpha 2-interferon (reaferon)

The use of electrochemical iodination under potentiostatic conditions resulted in generating 131I-labeled alpha 2-interferon (IF) the antiviral activity of which differed statistically insignificantly from that of the intact preparation. The resulting radioactive analogue of alpha 2-IF was successfully used for the study of pharmacokinetics of reaferon (RFN) in experimental animals which determined the parameters of the processes of its elimination from the blood stream, accumulation and release from the liver, the organ in which the highest accumulation of alpha 2-IF is observed. It is concluded from the foregoing that it would be expedient to use RFN for treatment of viral disease of the liver and the lungs.     

复制子荧光素酶表达质粒pSVK-luc的构建与应用

目的 为研究复制子DNA疫苗在生物活体内的表达情况,构建含有荧光素酶报告基因的复制子表达质粒pSVK-luc.方法 PCR扩增荧光素酶报告基因,克隆入DNA复制子载体pSVK,酶切鉴定和测序分析筛选阳性重组质粒pSVK-luc;化学法转染人胚肾293T细胞,流式细胞术和免疫荧光法检测荧光素酶基因在293T细胞中的表达;利用基因电穿孔导入仪递送重组质粒至BALB/c小鼠股四头肌,活体成像仪动态观测荧光素酶基因在小鼠体内的表达.结果 pSVK-luc经相应酶切和测序鉴定,与预期设计完全一致.流式细胞术和免疫荧光检测均可见荧光素酶基因表达;同时活体成像仪也能检测到该基因在小鼠体内的表达.结论 pSVK-luc表达质粒的构建成功将为复制子DNA疫苗体内作用机制研究和体内电穿孔递送条件的优化奠定实验基础.