接合转移质粒pUCP24T的构建与性能研究

目的 构建能在大肠埃希菌(E.coli)和铜绿假单胞菌(PA)间高效接合转移的质粒.方法 通过NCBI blastn程序分析质粒pCVD442接合反应起始序列oriT,PCR扩增oriT后插入pMD18-T,再将pMD1 8-oriT转化E.coli DH5α,经酶切、测序验证后用SmaI和HindIIII双酶切亚克隆至质粒pUCP24,获得质粒pUCP24T,将pUCP24T转化E.coli后研究pUCP24T从E.coli到PA的接合效率和质粒稳定性等.结果 成功构建pUCP24T重组质粒,在E.coli和PA的接合实验中,E.coli(pUCP24T)-PA共培养2h的接合效率平均为3.588×10-2,按12 h一代连续9代继代培养108 h,抗生素压力下质粒保存率为98.29％,无抗生素的培养基中质粒保存率为21.76％.结论 成功构建高接合效率的接合转移质粒pUCP24T.     

In vitro and in vivo transfection and expression of plasmid-based non-viral vector for erythropoietin gene therapy

A non-viral gene therapy vector, pcDNA3-EPO, was constructed by subcloning erythropoietin (EPO) cDNA into plasmid pcDNA3. After liposome-mediated transfection of the NIH 3T3 cells in vitro, EPO expression in the culture medium was detected by ELISA and amounted to 1.25 ± 0.3 IU ml^–1. The biological activity of this EPO in the medium was detected after intramuscular injection of BALB/c mice. PCR of genomic DNA and RT-PCR of total RNA also confirmed that the plasmid pcDNA3-EPO had been transfected into the cells. A pool of pcDNA3-EPO transfectants, which stably expressed EPO, was obtained by G418 selection. When pcDNA3-EPO was combined into liposomes and intramuscularly injected into BALB/c mice, the reticulocyte ratio in the positive mice was three times higher than that in the control mice. In vivo expression was maintained in mice for at least one month.     

大黄鱼源溶藻弧菌的鉴定及其菌蜕制备

【背景】菌蜕是诱导Phi X174噬菌体裂解基因E(Lysis E)在革兰氏阴性菌中表达后所获得无细胞内容物的细菌空壳。菌蜕生物安全性高,能以类似活菌方式诱导机体产生良好的系统和黏膜免疫应答。【目的】对分离自患溃疡病大黄鱼肝脏中的病原菌株16-3进行种属鉴定,利用温控调节表达系统控制Phi X174噬菌体裂解基因E在该菌株中的表达来制备菌蜕,为防控鱼类溶藻弧菌感染提供有效手段。【方法】采用形态特征观察、生理生化特性测定及16S r RNA基因序列分析等方法对菌株16-3进行鉴定;构建温控裂解质粒p BV220-Lysis E,并将其电转至溶藻弧菌菌株16-3,形成重组溶藻弧菌菌株16-3(p BV220-Lysis E);将不同起始浓度的重组溶藻弧菌培养物同时进行42°C升温诱导,比较其溶菌动力曲线和裂解效率的差异;在最佳条件下制备溶藻弧菌菌株16-3菌蜕,电镜观察其形态与结构,采用倾注平板法测定冻干菌蜕中的活菌数。【结果】综合菌株16-3在形态、生理生化及16S r RNA基因系统发育等方面的特性,确定其为溶藻弧菌;构建了温控裂解质粒p BV220-Lysis E和重组溶藻弧菌菌株16-3(p BV220-Lysis E);溶藻弧菌菌株16-3菌蜕制备的最佳条件是选择起始浓度OD600为0.3的菌液进行诱导,诱导3 h后即可收获菌蜕,其裂解效率为96.9%,但经冻干处理后的菌蜕无活菌残留;电镜观察发现菌株16-3菌蜕保持原细胞的基本形态,但细胞表面有明显的溶菌孔道,且由于细胞内容流失而使细胞表面发生皱缩。【结论】制备出溶藻弧菌菌株16-3菌蜕,为其作为疫苗或疫苗递送载体奠定了基础。     

ATPase activity of SopA, a protein essential for active partitioning of F plasmid

Summary The SopA, B, C genes of the F plasmid play an essential role in plasmid partitioning during cell division in Escherichia coli. In this paper, the products of the sopA and sopB genes were isolated and their biochemical activities studied. [-³²P]ATP was cross-linked to the SopA protein by UV irradiation; this cross-linking was observed only in the presence of magnesium ion, and was competitively inhibited in the presence of non-radioactive ATP, ADP and dATP, but not other NTPs or dNTPs. In contrast, no ATP binding activity was detected for the SopB protein. The SopA protein showed a modest magnesium ion-dependent ATPase activity and this activity was stimulated in the presence of DNA. The ATPase activity in the presence of DNA was further stimulated by addition of the SopB protein. However, the SopB protein alone failed to stimulate the ATPase activity.     

Evidence for a life span-prolonging effect of a linear plasmid in a longevity mutant of Podospora anserina

The linear mitochondrial plasmid pAL2-1 of the long-lived mutant AL2 of Podospora anserina was demonstrated to be able to integrate into the high molecular weight mitochondrial DNA (mtDNA). Hybridization analysis and densitometric evaluation of the mitochondrial genome isolated from cultures of different ages revealed that the mtDNA is highly stable during the whole life span of the mutant. In addition, and in sharp contrast to the situation in certain senescence-prone Neurospora strains, the mutated P. anserina mtDNA molecules containing integrated plasmid copies are not suppressive to wild-type genomes. As demonstrated by hybridization and polymerase chain reaction (PCR) analysis, the proportion of mtDNA molecules affected by the integration of pAL2-1 fluctuates between 10% and 50%. Comparative sequence analysis of free and integrated plasmid copies revealed four differences within the terminal inverted repeats (TIRs). These point mutations are not caused by the integration event since they occur subsequent to integration and at various ages. Interestingly, both repeats contain identical sequences indicating that the mechanism involved in the maintenance of perfect TIRs is active on both free and integrated plasmid copies. Finally, in reciprocal crosses between AL2 and the wild-type strain A, some abnormal progeny were obtained. One group of strains did not contain detectable amounts of plasmid pAL2-1, although the mtDNA was clearly of the type found in the long-lived mutant AL2. These strains exhibited a short-lived phenotype. In contrast, one strain was selected that was found to contain wild-type A-specific mitochondrial genomes and traces of pAL2-1. This strain was characterized by an increased life span. Altogether these data suggest that the linear plasmid pAL2-1 is involved in the expression of longevity in mutant AL2.     

The goldfish hAT-family transposon Tgf2 is capable of autonomous excision in zebrafish embryos

The goldfish (Carassius auratus) Tgf2 transposon is a vertebrate DNA transposon that belongs to the hAT transposon family. In this study, we constructed plasmids containing either the full-length Tgf2 transposon (pTgf2 plasmid) or a partially-deleted Tgf2 transposon (ΔpTgf2 plasmid), and microinjected these plasmids into fertilized zebrafish (Danio rerio) eggs at the one- to two-cell stage. DNA extracted from the embryos was analyzed by PCR to assess transient excision, if any, of the exogenous plasmid and to verify whether Tgf2 is an autonomous transposon. The results showed that excision-specific bands were not detected in embryos injected with the ΔpTgf2 plasmid, while bands of 300–500 bp were detected in embryos injected with pTgf2, which indicated that the full-length Tgf2-containing plasmid could undergo autonomous excision in zebrafish embryos. DNA cloned from 24 embryos injected with pTgf2 was sequenced, and the results suggested that Tgf2 underwent self-excision in zebrafish embryos. Cloning and PCR analysis of DNA extracted from embryos co-injected with ΔpTgf2 and in vitro-transcribed transposase mRNA indicated that partially-deleted-Tgf2-containing ΔpTgf2 plasmid also underwent excision, in the presence of functional transposase mRNA. DNA cloned from 25 embryos co-injected with ΔpTgf2 and transposase mRNA was sequenced, and the results suggested that partially-deleted Tgf2 transposons plasmids were excised. These results demonstrated that excisions of Tgf2 transposons were mediated by the Tgf2 transposase, which in turn confirmed that Tgf2 is an autonomous transposon.     

Conjugative transfer of plasmid pTd33 in agrobacteria

Supramembrane structures that connect conjugating agrobacterial cells were visualized for the first time by transmission electron microscopy. The primary contact of cells during conjugation was shown to occur through the formation of long pili containing no VirB1 protein. Pretreatment of agrobacterial cells with acetosyringone resulted in a six-to tenfold increase in the transfer frequency of plasmid pTd33 at 19–25°C and had almost no effect at 30°C. The transfer of plasmid pTd33 fromA. tumefaciens strain GV3101 to plasmid-freeA. tumefaciens strain UBAPF-2 was 16 times decreased after the centrifugation of cells. The transfer efficiency of plasmid pTd33 fromA. tumefaciens strain LBA2525 (virB2::lacZ) to plasmid-freeA. tumefaciens strain UBAPF-2 was one order of magnitude lower than the transfer from the wild-typeA. tumefaciens strain GV3101. Treatment of donor cells with 0.01% SDS before mating decreased the transfer efficiency by a factor of 26. The role of pili in the establishment of contact between conjugating cells of agrobacteria is discussed.     

The tricarboxylic acid cycle in L₃ Teladorsagia circumcincta: metabolism of acetyl CoA to succinyl CoA

Nematodes, like other species, derive much of the energy for cellular processes from mitochondrial pathways including the TCA cycle. Previously, we have shown L₃Teladorsagia circumcincta consume oxygen and so may utilise a full TCA cycle for aerobic energy metabolism. We have assessed the relative activity levels and substrate affinities of citrate synthase, aconitase, isocitrate dehydrogenase (both NAD⁺ and NADP⁺ specific) and α-ketoglutarate dehydrogenase in homogenates of L₃T. circumcincta. All of these enzymes were present in homogenates. Compared with citrate synthase, low levels of enzyme activity and low catalytic efficiency was observed for NAD⁺ isocitrate dehydrogenase and especially α-ketoglutarate dehydrogenase. Therefore, it is likely that the activity of these to enzymes regulate overall metabolite flow through the TCA cycle, especially when [NAD⁺] limits enzyme activity. Of the enzymes tested, only citrate synthase had substrate affinities which were markedly different from values obtained from mammalian species. Overall, the results are consistent with the suggestion that a full TCA cycle exists within L₃T. circumcincta. While there may subtle variations in enzyme properties, particularly for citrate synthase, the control points for the TCA cycle in L₃T. circumcincta are probably similar to those in the tissues of their host species.     

Control of cloned gene expression by promoter inversion in vivo: construction of the heat-pulse-activated att-nutL-p-att-N module

We have constructed a prototype of gene-expression plasmids with three novel properties: its "OFF phase" is absolute in all common hosts because the expression promoter is facing away from the studied gene and is blocked by a strong terminator; the "ON phase" is attained by the rapid and efficient inversion of the promoter; only a short heat pulse or exposure to other inducing agents is required to initiate this two-stage process. In the first stage, synthesis of the phage lambda Int protein is induced by the transient derepression of the properly engineered lambda xis- cIts857 prophage. In the immediately following second stage, Int causes inversion of a promoter cloned between the inverted ----P'OP phage att site and the normally oriented ----delta PO delta P' pseudo-bacterial att site. The inverted promoter can now control the expression of the studied gene and also of the lambda N gene cloned in tandem. The N product, in conjunction with the nutL site placed downstream of the promoter, permits efficient antitermination of any terminators present in the att sites, in the plasmid or in the cloned DNA, making this system efficient and of practical value. Employing the promoter-inverting plasmid, it was possible to obtain rapid onset and a high level of galactokinase synthesis from the cloned galK gene. Only a transient, 10-min induction at 42 degrees C was employed, permitting protein synthesis at 30 degrees C, which might be of importance for thermosensitive products. Furthermore, the entire promoter-inversion module can be transferred to any plasmid as a 1.3-kb AvaI-ClaI fragment (see Fig. 1).(ABSTRACT TRUNCATED AT 250 WORDS)