异源MAR介导外源基因与家蚕受精卵核基质的体外结合

以携带人β干扰素基因5′上游MAR(Matrix Association regions)的质粒pCL为载体, 在其BamHI位点上正向插入天蚕(Antheraea ramamai)丝素基因核心区Af6.8而构建成质料pMAC。通过质粒pMAC、pAy6.8和pCL与家蚕(Bombyxmori)受精卵核基质的体外结合及其比较分析发现,在体外异源MA R能有效地介导外源基因与核基质结合。这为MAR作为真核基因转移、整合和表达的载体提供了一个初步的实验证据。 Abstract:We have constructed a plasmid pMAC by inserting the core region of Antheraen yamamai fibroin gene into the BamHI site of the plasmid pCL which contains the MAR upstream huIFN-βgene.In vitro association of pCL,pAy6.8 and pMAC with the nuclear matrix of Bombyx mori fertilized eggs indicated that pMAC and pCL could tightly bind to the nuclear matrix,but pAy 6.8 found much more ineffectively.This provides a preliminary evidence for MAR as a vector for eukaryotic genes tranfer,integration and expression.     

Complementation analysis of the aliphatic amidase genes of Pseudomonas aeruginosa

A plasmid, pCL34, capable of autonomous replication in Escherichia coli and Pseudomonas aeruginosa has been constructed which carries the promoter and structural gene (amiE) for P. aeruginosa amidase, but not the regulator gene (amiR). Plasmid pCL34 has been mobilized from E. coli to P. aeruginosa using the broad host range plasmid RP4. Complementation studies were performed in P. aeruginosa strains carrying various amidase mutations. Measurements of amidase activity in the recipients under inducing, non-inducing and repressing conditions showed trans-complementation by the chromosomally located regulator gene product. These results confirmed the positive control model for amidase gene expression. Levels of amidase expression seen during these studies were approximately threefold higher than in the parental, amidase-positive strains.     

Maintenance of pBR322-derived plasmids without functional RNAI

pBR322-derived plasmids that lack the bla gene and 40% of the gene for the replication inhibitor, RNAI, have been constructed. Since the RNAI gene totally overlaps with the gene for the replication primer, RNAII, this primer is similarly defective and also lacks its normal promoter. The primer is presumed to by synthesized either from the counter-tet promoter (plasmid pCL59) or from an inserted lacUV5 promoter (plasmid pCL59-65). Based mainly on the observation that the plasmid Rom protein, which normally assists in the RNAI/RNAII interaction, has no effect on the replication of the RNAI/RNAII-defective plasmids, we suggest that the defective RNAI is not functional while the defective RNAII primer, although less efficient, still allows plasmid replication. The defective plasmids are fully compatible with the intact parent plasmid, indicating that they do not share a common control of replication. In the absence of antibiotics, the bacteria lose the defective plasmid, beginning after 80 generations; under the same conditions, the parent plasmid is retained even after 140 generations. During exponential growth of their host, the number of defective plasmids in a culture increases exponentially with a doubling time either smaller or greater than that of the host cell growth, depending on the growth medium and, in the case of pCL59-65, on the presence or absence of lac inducer IPTG. As a result of these differences in host cell growth and plasmid replication, the plasmids are either gradually diluted out or their copy number continually increases. This shows that, without RNAI, plasmid replication is uncoupled from the host cell growth and not, as usual, adjusted to it. It also implies that the RNAI mechanism is the only means of replication control for ColE1-type plasmids that senses and adjusts the copy number; limiting host factors cannot provide a back-up control to stabilize copy numbers.     

Expression of organophosphate hydrolase in the filamentous fungus Gliocladium virens

The broad-spectrum organophosphate hydrolase (OPH; EC 3.1.8.1) encoded by the organophosphate-degrading gene (opd) from Pseudomonas diminuta MG and Flavobacterium sp. ATCC 27551 possesses capabilities of both P-O bond hydrolysis (e.g. paraoxon) and P-F bond hydrolysis [e.g. sarin and diisopropylfluorophosphate (DFP)]. In the present study a 9.4-kb plasmid, pCL1, was used to transform the saprophytic fungus Gliocladium virens. pCL1 was derived from pJS294 by placing the fungal promoter (prom1) from Cochliobolus heterostrophus upstream and the trpC terminator from Aspergillus nidulans downstream of the opd gene. Southern analysis of restricted genomic DNA from various transformants indicated that integration occurred non-specifically at multiple sites. Western blot analysis of mycelial extracts from transformants confirmed the production of a processed form of the enzyme in the fungus. Maximal levels of OPH activity (rate of p-nitrophenol production from paraoxon) were observed after 168 h of culture and activity levels correlated with biomass production in mature vegetative growth.     

Plasmid and serological differences between Edwardsiella ictaluri strains.

Several studies have shown that isolates of Edwardsiella ictaluri obtained from infected channel catfish in the southeastern United States harbor two cryptic plasmids, designated pCL1 (5.7 kb) and pCL2 (4.9 kb). These isolates appear to be serologically homogeneous. To extend these studies, we focused our analyses on two isolates of nonictalurid origin. Plasmid analyses of a danio isolate showed that it harbored plasmids which were similar if not identical to pCL1 and pCL2. This strain was also serologically indistinguishable from those isolated from channel catfish. In contrast, a green knife fish (GNF) isolate harbored four plasmids with relative mobilities of 6.0, 5.7, 4.1, and 3.1 kb. Southern blot analyses indicated that only the 5.7- and 4.1-kb plasmids strongly hybridized under high-stringency conditions to probes specific for pCL1 and pCL2, respectively. The GNF isolate showed minimal reactivity when reacted with polyclonal antiserum prepared against a channel catfish isolate. However, polyclonal antiserum to the GNF isolate strongly reacted with the GNF isolate in both surface fluorescence and agglutination reactions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of cell lysates showed that the protein banding patterns of the strains compared were similar. However, Western blots of proteinase K-digested cell extracts showed that O antigen of the GNF isolate was antigenically distinct from the O antigen of the other isolates. These studies indicate that there are different serotypes of E. ictaluri and suggest that plasmid and serological analyses of future isolates of E. ictaluri can be used to determine whether structurally distinct strains are emerging in major channel catfish aquaculture areas.     

Characterization of a high molecular mass resistance plasmid isolated from Klebsiella pneumoniae and coding for lactose degradation

Abstract Originated from Klebsiella pneumoniae , pCL300 is a self-transmissible plasmid, coding for multiple antibiotic resistances and for lactose degradation. This fi⁺ plasmid belongs to the incompatibility group F I and is stably maintained in various lactose-negative Escherichia coli or in Salmonella typhi . The molecular mass determined by agarose gel electrophoresis after endonuclease digestions and confirmed by the electron microscope was 243 ± 15 kb, an unusually high value for a plasmid belonging to the incF I group.     

Construction and functional characteristics of tuber-specific and cold-inducible chimeric promoters in potato

The improvement of processing quality of potato products (fries and chips) demands less accumulation of reducing sugars (glucose and fructose) in cold-stored potato (Solanum tuberosum) tubers. Control of gene expression to achieve this requires promoters with specificity to tubers as well as inducible activity under low temperatures. Here we use overlapping extension PCR to construct two chimeric promoters, pCL and pLC, to control gene expression in a tuber-specific and cold-inducible pattern. This combined different combinations of the LTRE (low-temperature responsive element) from Arabidopsis thaliana cor15a promoter and the TSSR (tuber-specific and sucrose-responsive sequence) from potato class I patatin promoter. The cold-inducible and tuber-specific activities of the chimeric promoters were investigated by quantitative analysis of GUS activity in transgenic potato cultivar E3 plants. The results showed that the cis-elements, LTRE and TSSR, played responsive roles individually or in combination. pCL with the TSSR closer to the TATA-box showed substantially higher promoter activity than pLC with the LTRE closer to the TATA-box at either normal (20°C) or low temperature (2°C), suggesting that the promoter activity was closely associated with the position of the two elements. The chimeric promoter pCL with tuber-specific and cold-inducible features may provide valuable tool for controlling the expression of gene constructs designed to lower the formation of reducing sugars in tubers stored at low temperature and to improve the processing quality of potato products. The nucleotide sequence data reported will appear in the GenBank database under the accession numbers DQ494557 (pCL) and DQ494558 (pLC ).     

Exploration of critical parameters for transient retrovirus production

The pCL system was developed to aid in the production of retrovirus that encodes cytotoxic or cytostatic cDNA's. A principal feature of this system is the transient production of virus after co-transfection of the viral and packaging vectors in the 293T cell line. This approach obviates the need for selection of the producer cells, thus minimizing potential affects of the encoded genes. However, the transient nature of this system also creates a number of experimental variables. In this study we have examined and optimized elements related to the production of the pCL retrovirus. For example, co-transfection of the packaging sequence along with the viral vector has been optimized in terms of both the total amount of DNA transfected and the relative proportion of each plasmid. We have also tested the affect of increased synthesis of viral proteins in the producer cells and the kinetics of virus accumulation in the supernatant. These findings may be of interest to those who use pCL or any transient packaging system in their gene transfer studies. In addition, these studies may aid in the validation and development of transient retrovirus production systems for clinical applications.     

A gene coding for 3-deoxy-D-manno-octulosonic-acid transferase in Escherichia coli. Identification, mapping, cloning, and sequencing

An autoradiographic assay applicable to colonies immobilized on filter paper was developed for obtaining temperature-sensitive mutants of Escherichia coli defective in the transfer of 3-deoxy-D-manno-octulosonic acid (KDO) from CMP-KDO to a tetraacyldisaccharide 1,4'-bisphosphate precursor of lipid A, designated lipid IVA. Cell-free extracts from two mutants found in a population of 30,000 mutagen-treated cells showed normal KDO transferase activity when assayed at 30 degrees C, but almost no activity at 42 degrees C. The mutation was mapped by mating one of the mutants with different Hfr strains and analyzing genetic linkage of KDO transferase activity to selectable markers. The lesion was located to a position between 80 and 84 min on the E. coli chromosome. A plasmid from the Clarke and Carbon collection (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99), pLC17-24, known to contain genes from the rfa region (81 min), was shown to overexpress KDO transferase activity 4-5 times and to correct the mutation when the plasmid was conjugated into the mutant strains. The KDO transferase gene, designated kdtA, was subcloned from pLC17-24 into a multicopy vector. The resulting plasmid, pCL3, overproduced transferase activity approximately 100-fold. The kdtA gene was shown to code for a 43-kDa polypeptide, as judged by radiolabeling of minicells. Its DNA sequence was determined. The results demonstrate that overexpression of this single gene product greatly stimulates the incorporation of two stereochemically distinct KDO residues during lipopolysaccharide biosynthesis in extracts of E. coli.     

Isolation and purification of propionicin PLG-1, a bacteriocin produced by a strain of Propionibacterium thoenii.

Production of propionicin PLG-1 by Propionibacterium thoenii P127 was pH dependent, with maximal activity detected in supernatants of cultures grown at pH 7.0 Propionicin PLG-1 was purified by ion-exchange chromatography and isoelectric focusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of propionicin PLG-1 purified through isoelectric focusing resolved a protein band with a molecular weight of 10,000. Propionicin PLG-1 was bactericidal to sensitive cells, demonstrating single-hit kinetics. The producing strain harbored a single plasmid (pLG1) with an approximate size of 250 kb. Preliminary data indicate that both propionicin PLG-1 and immunity to the bacteriocin are encoded on the chromosome. Exposure of strain P127 to acriflavine or to N-methyl-N'-nitro-N-nitrosoguanidine yielded isolates that no longer produced bacteriocin activity and isolates that were cured of the plasmid. However, loss of bacteriocin production was not correlated with loss of the plasmid. Isolates cured of the plasmid were phenotypically identical to plasmid-bearing cells in fermentation patterns, pigment production, and growth characteristics.     

Constructing a test system for the identification of plague microbe based on genetic probes]

DNA probes for detection of the plague agent Yersinia pestis were made on a basis of its three typical extrachromosomal replicons. The recombinant plasmid pBS2 including pBR327 vector and SalGI-BspRI fragment of the plasmid pFra was constructed. The above fragment is connected with synthesis of Y. pestis capsular antigen and it is a 400 bp species-specific DNA probe called F1 which is suitable for identification of Y. pestis species that bears the 60 mdal plasmid. The DNA probes called P1 was made on a basis of the plasmid pPst; it is the 460 BglII-BamHI fragment of the fibrinolysin-coagulase gene suitable for species-specific detection of Y. pestis species that bears the 60 mdal plasmid. The P1 fragment was cloned into the pAT153 vector and the constructed recombinant plasmid was called pEK7. The recombinant plasmid pCL1, including the pBR325 vector and the 6th BamHI fragment of Y. pestis EV plasmid pCad was constructed. The above fragment includes the replication origin of the pCad and it is hybridized to the pCad-bearing strains of Y. pestis and Y. tuberculosis only. Thus, it may be a basis for a bi-species-specific DNA probe making. These three recombinant plasmids are considered as a test-system for detection of both typical and atypical strains of Y. pestis.     

Plasmid Control of 6-Aminohexanoic Acid Cyclic Dimer Degradation Enzymes of Flavobacterium sp. K172

Flavobacterium sp. K172, which is able to grow on 6-aminohexanoic acid cyclic dimer as the sole source of carbon and nitrogen, and plasmid control of the responsible enzymes, 6-aminohexanoic acid cyclic dimer hydrolase and 6-aminohexanoic acid linear oligomer hydrolase, were studied. The wild strain of K172 harbors three kinds of plasmid, pOAD1 (26.2 megadaltons), pOAD2 (28.8 megadaltons), and pOAD3 (37.2 megadaltons). The wild strain K172 was readily cured of its ability to grow on the cyclic dimer by mitomycin C, and the cyclic dimer hydrolase could not be detected either as catalytic activity or by antibody precipitation. No reversion of the cured strains was detected. pOAD2 was not detected in every cured strain tested but was restored in a transformant. The transformant recovered both of the enzyme activities, and the cyclic dimer hydrolase of the transformant was immunologically identical with that of the wild strain. All of the strains tested, including the wild, cured, and transformant ones, possessed identical pOAD3 irrespective of the metabolizing activity. Some of the cured strains possessed pOAD1 identical with the wild strain, but the others harbored plasmids with partially altered structures which were likely to be derived from pOAD1 by genetic rearrangements such as deletion, insertion, or substitution. These results suggested that the genes of the enzymes were borne on pOAD2.     

Evidence for plasmid mediated dissimilation of catechol in Azotobacter chroococcum

Catechol dissimilation in A. chroococcum was encoded by plasmid pMSB1. This self transmissible plasmid was effectively cured by mitomycin C. The cured cells did not harbour the plasmid and failed to utilize catechol.     

Study of two bacteriocins produced by Enterococcus casseliflavus and Ent. faecalis

AIMS: The antimicrobial activity of two plasmid-borne bacteriocins produced by Enterococcus casseliflavus IM 416K1 and Ent. faecalis IM 388C and their mating transferability were studied. METHODS AND RESULTS: Both bacteriocins showed antibacterial activity against taxonomically related micro-organisms and Listeria monocytogenes but differ for heat sensitivity, antimicrobial titre, molecular size and class of affiliation. The transferability by mating of the antibacterial properties from producers to Enterococcus faecalis JH2-2 revealed that the bacteriocin-phenotype was linked in both strains to genes located on a 34 MDa plasmid. This result was confirmed by loss of antibacterial activity and immunity after curing treatment. CONCLUSIONS: Restriction analysis has shown a different profile of the two conjugative plasmids. Enterocin 416K1 and Enterocin 388C could represent natural antilisterial agents to use in food technology. SIGNIFICANCE AND IMPACT OF THE STUDY: The transferability of the 34 MDa conjugative plasmids might be considered a possibility for the study of bacteriocins expression in bacterial hosts different from the native strains.     

眼镜蛇变形杆菌病的病原鉴定及治疗

目的为查明发病蛇的病原以及提供有效的治疗措施。方法用营养琼脂、鲜血琼脂、SS培养基分离培养病原菌,并进行生化试验和体外抑菌试验。结果从病原菌的培养特性、菌落形态和生化试验结果可以确定为普通变形杆菌,体外抑菌试验结果表明病原菌对阿奇霉素、恩诺沙星等抗菌药高度敏感。结论用高度敏感抗菌药治疗病蛇,效果显著。     

鸽β-防御素1基因的克隆及其生物学作用鉴定

【目的】从鸽子组织中克隆鸽子β-防御素1(AvBD1)基因,在大肠杆菌中表达重组鸽子AvBD1蛋白,测定其生物学特性。【方法】应用RT-PCR法从鸽子骨髓组织中扩增鸽子AvBD1基因,采用Real-time PCR法检测该基因在鸽子组织器官中的表达分布。将该基因亚克隆到大肠杆菌原核表达载体pProEX-HTa的EcoR I和Xho I双酶切位点上,构建重组表达质粒pProEX-pigeon AvBD1,将重组质粒进行诱导表达;对该重组蛋白进行纯化,通过菌落计数法测定其体外抗菌活性与理化特性。【结果】从鸽子骨髓组织中克隆到鸽子AvBD1基因,其cDNA大小为198 bp,编码65个氨基酸,经序列相似性分析,鸽子AvBD1与鸭AvBD1氨基酸序列相似性最高(81.5%)。鸽子AvBD1主要分布于免疫系统和消化系统组织中。Tricine-SDS-PAGE电泳结果表明,重组鸽子AvBD1蛋白分子量约8.8 kD,与预期大小一致。该重组蛋白具有广谱抗菌活性,高盐浓度显著降低其抗菌活性。此外,该重组蛋白的溶血活性极低。【结论】从鸽子骨髓组织中克隆到鸽子AvBD1基因,其主要分布在机体的免疫系统和消化系统中。该重组蛋白具有广谱抗菌活性,高盐浓度显著降低其抗菌活性,且该重组蛋白的溶血活性极低。     

The molecular design of a recombinant antimicrobial peptide CP and its in vitro activity

Antibacterial peptides from various sources express different antibacterial activity. In order to obtain a high activity antibacterial peptide, the sequences of four antimicrobial peptides--Protegrin-1, 4 kDa Scorpion Defensin, Metalnikowin-2A and Sheep Myeloid Antibacterial Peptide SMAP-29--were exploited to generate a synthetic antimicrobial peptide cp gene, which was then cloned into the expression vector pPICZalpha-A. The constructed recombinant expression vector pPICZalpha-cp was transformed into Pichia pastoris X-33, in which the synthetic antimicrobial peptide (CP) could be expressed under the control of the inducible AOX1 promoter and secreted via the alpha mating factor leader of Saccharomyces cerevisiae. Results showed that recombinant plasmid is highly stable, and In vitro experiments showed that the recombinant antimicrobial peptide CP is heat and acid-stable, and it has high antibacterial activity against several Gram-positive and -negative bacteria. Only 1 microg of the recombinant antimicrobial peptide CP has an antibacterial activity equivalent to 64 U ampicillin. Thus, this recombinant antimicrobial peptide could serve as an attractive candidate for the development of therapeutic antimicrobial drugs.     

Antibacterial and antidiarrhoeal effects of alkaloids of Holarrhena antidysenterica WALL

The alkaloids from the ethanolic extract of H. antidysenterica seeds were evaluated for their antibacterial activity against clinical isolates of enteropathogenic Escherichia coli (EPEC) in vitro, and their antidiarrhoeal activity on castor oil-induced diarrhoea in rats, in vivo. The plasmid DNA, whole cell lysate and outer membrane protein profile of a clinical isolate of EPEC was determined in presence of alkaloids of H. antidysenterica. The disc diffusion and agar well diffusion methods were used to evaluate the antibacterial efficacy. The alkaloids showed strong antibacterial activity against EPEC strains. In castor oil-induced diarrhoea, alkaloids reduced the diarrhoea with decrease in the number of wet faeces in pretreated rats at a dose of 200-800 mg/kg. The loss of plasmid DNA and suppression of high molecular weight proteins were observed on alkaloids treatment. Taking into account the multiple antibiotic resistance of EPEC, the results suggest usefulness of alkaloids of H. antidysenterica seeds as antibacterial and antidiarrhoeal agents.     

Plasmid-mediated serum resistance and alterations in the composition of lipopolysaccharides in Salmonella dublin

Survival rates of Salmonella dublin in rabbit serum after culture for 1 h at 37 degrees C were compared between a wild-type strain (5240) carrying a 50 MDa plasmid, a plasmid-cured strain (C524), and a cured strain containing the 50 MDa plasmid tagged with Tn1 (5241). Strain C524 was more susceptible to the bactericidal activity of normal serum than its parent strain 5240 (percentage survival less than 1% and 52.5 +/- 9.2%, respectively). On the other hand, the percentage survival of strain 5241 was significantly increased (90.4 +/- 4.0%), indicating that the reintroduction of the plasmid into the cured strain restored the serum resistance. Moreover, this change in the serum resistance properties correlated with changes in the neutral sugar composition of the lipopolysaccharides (LPS) of these strains, suggesting that the 50 MDa plasmid is necessary for O-side chain expression in the LPS of S. dublin.