Plasmids for recombination-based screening.

To facilitate recombination-based screening, we constructed the ColE1-based plasmid, pi G4, that confers chloramphenicol resistance, contains a polylinker with multiple unique restriction enzyme recognition sequences, and contains the genetic marker, supF. To facilitate recombination-based screening followed by rapid DNA sequencing, we inserted the selectable marker, supF, into each of 20 high-copy-number (hcn) pUC-derived NoC plasmids that were designed for multiplex DNA sequencing. To facilitate recombination-based screening of common cDNA libraries that often contain ColE1 sequences, we constructed a supF-carrying plasmid whose replication was driven from an R6K replicon that does not share sequence homology with ColE1. Furthermore, we incorporated a useful polylinker and increased the copy number of this plasmid to create the 4.4-kb hcn plasmid, pMAD1. Thus, these plasmids allow: (1) background-free transformation of cells by a supF plasmid carrying an antibiotic-resistance marker; (2) simultaneous performance of the recombination-based assay and DNA sequencing; and (3) screening bacteriophage cDNA libraries that contain ColE1 sequences by recombination with a supF plasmid that is not homologous to ColE1 derivatives.     

Shuttle cloning vectors for the cyanobacterium Anacystis nidulans.

Hybrid plasmids capable of acting as shuttle cloning vectors in Escherichia coli and the cyanobacterium Anacystis nidulans R2 were constructed by in vitro ligation. DNA from the small endogenous plasmid of A. nidulans was combined with two E. coli vectors, pBR325 and pDPL13, to create vectors containing either two selectable antibiotic resistance markers or a single marker linked to a flexible multisite polylinker. Nonessential DNA was deleted from the polylinker containing plasmid pPLAN B2 to produce a small shuttle vector carrying part of the polylinker (pCB4). The two polylinker-containing shuttle vectors, pPLAN B2 and pCB4, transform both E. coli and A. nidulans efficiently and provide seven and five unique restriction enzyme sites, respectively, for the insertion of a variety of DNA fragments. The hybrid plasmid derived from pBR325 (pECAN1) also transforms both E. coli and A. nidulans, although at a lower frequency, and contains two unique restriction enzyme sites.     

Apparent incompatibility of plasmid pSfrYC4b of <Emphasis Type="Italic">Sinorhizobium fredii</Emphasis> with two different plasmids in another strain

Sinorhizobium fredii YC4B is a spontaneous mutant derivative of strain YC4 that is unable to nodulate soybeans. The second-largest plasmid of strain YC4B, termed pSfrYC4b (810 kb), was transferred to S. fredii HN01SR, a strain which contains three large indigenous plasmids (pSfrHN01a, pSfrHN01b and pSfrHN01c). Surprisingly, two stable indigenous plasmids (pSfrHN01a and pSfrHN01b) of strain HN01SR were cured simultaneously by the introduction of pSfrYC4b. Furthermore, a novel, unstable plasmid (pHY4) became visible in agarose gels. The electrophoretic mobility of plasmid pHY4 was slower than that shown by the cured plasmids, indicating that the molecular weight of the former is higher than that of plasmids pSfrYC4b and pSfrHN01b. Replication gene repC-like sequences were detected by polymerase chain reaction (PCR) on pSfrHN01a and pSfrYC4b, but not on pSfrHN01b. Sau3AI and PstI restriction patterns of the PCR-amplified repC-like sequences from HN01SR and YC4B were very similar.     

Establishment of a novel sensitive method for detecting methylation modification on DNA of escherichia coli cell

This study focused on finding a novel sensitive method to determine the methylation modification at DNA dam (GATC) sites in Escherichia coli. A new plasmid which contained three GATC sites recognized by restriction enzyme BclI and one GAATTC site recognized by EcoRI was transformed into E. coli stains AB1157(dam ⁺) and GM2929(dam ⁻) respectively. Then the plasmid DNA was digested by restriction enzyme BclI(T*GATCA), which was sensitive to methylation. The results showed that the plasmid derived from AB1157 was not digested while that from GM2929 was, for the methylation level of the former was high while the latter was low. So by detecting the methylation of plasmid transferred into the strain, we could determine whether methylaion existed at DNA dam (GATC) site in E. coli. This method was effective and rapid; moreover, the digested fragments were not dispersive. It also made a basis for the detection of whether methylation occurred in mode beings by low-energy ion beam. The article is published in the original.     

A novel shuttle cloning vector for the cyanobacterium Anacystis nidulans

Abstract Shuttle cloning vectors for use with the cyanobacterium Anacystis nidulans and Escherichia coli were constructed by combining an endogenous A. nidulans plasmid with an E. coli vector containing a 14 site non-symmetrical polylinker. The resulting plasmids, designated pPLAN B1 and pPLAN B2, transform A. nidulans with high efficiency and contain 7 unique restriction enzyme sites suitable for cloning.     

pFC1 to pFC7: A novel family of combinatorial cloning vectors

We have designed and constructed a series of plasmid vectors based on pBlueScript, where additional restriction sites have been incorporated into theSstI andKpn I sites. These sites, of enzymes that cut only rarely, permit expression cassettes constructs to be easily excised and multimerized with others.     

A set of novel Ti plasmid-derived vectors for the production of transgenic plants

Summary We describe in this paper the construction and use of a set of novel Ti plasmid-derived vectors that can be used to produce transgenic plants. These vectors are based on one of two strategies: 1) double recombination into the wild-type Ti plasmid of genetic information flanked by two T-DNA fragments on a wide-host range plasmid; 2) the binary vector strategy. The vector based on the double recombination principle contains a kanamycin resistance gene for use as a plant selectable marker, a polylinker for the insertion of foreign genes, and a nopaline synthase gene. The vector was constructed such that a disarmed T-DNA results from the double recombination event. The binary vector combines several advantageous features including an origin of replication that is stable in Agrobacterium in the absence of selection, six unique sites for insertion of foreign genes, an intact nopaline synthase gene, and a kanamycin resistance marker for selection of transformed plant cells. All of these vectors have been used to produce tobacco plants transformed with a variety of foreign genes.     

菠菜甜菜碱醛脱氢酶基因在烟草中的表达

质粒pLS9含有1．5kb的编码菠菜甜菜碱醛脱氢酶(BADH)基因。经限制酶切后克隆到植物表达载体的35S启动子和PolyA终止子之间。经农杆菌介导转化烟草,获得90多株抗卡那霉素再生植株。经PCR检测证明60％以上再生植株含有BADH基因。转基因植株经Western blot,BADH酶活性测定,BADH酶活性特异性染色法检查和耐盐性分析,证明菠菜BADH基因在烟草正常表达。在叶绿体和胞液中均有BADH酶存在。转基因植株能耐较高浓度盐。     

小鼠白细胞介素33真核表达质粒的构建及表达

克隆小鼠IL-33基因构建其真核表达质粒,并转染COS-7细胞检测其表达。提取C57BL/6小鼠肺组织总RNA,经反转录聚合酶链式反应（RT-PCR）扩增小鼠IL-33基因,酶切后插入pcDNA^TM3.1/myc HisA构建其真核表达质粒pcDNA-3.1-IL-33,重组质粒转染COS-7细胞,RT-PCR和免疫印迹法（western blotting）检测目的基因表达。结果显示,pcDNA3.1-IL-33中插入的片段序列测定结果与小鼠IL-33cDNA序列一致,重组质粒转染COS-7细胞后检测到相应mRNA及蛋白表达。成功克隆了小鼠IL-33基因cDNA,并构建其真核表达质粒。     

Mapping and genetic organization of pTiChry5, a novel Ti plasmid from a highly virulent Agrobacterium tumefaciens strain

Agrobacterium tumefaciens Chry5, a wild-type strain originally isolated from chrysanthemum, is unusually tumorigenic, particularly on soybean. We have mapped the Chry5 Ti plasmid by genomic walking and restriction endonuclease analysis, and have located its virulence, T-DNA, plasmid incompatibility, and l,l-succinamopine utilization loci. Southern analysis has revealed that about 85% of the Chry5 Ti plasmid is highly homologous to another Ti plasmid, pTiBo542. Although all the functions that we have located on pTiChry5 are encoded by pTiBo542-homologous regions, the two Ti plasmids differ in their genetic organization. The overall patterns of restriction sites in the plasmids also differ, with the exception of an approximately 12 kb segment of the virulence region, where the BamHI sites appear to be conserved. Complementation analysis has shown that deletion of a DNA segment which flanks the oncogenic T-DNA results in severe attenuation of virulence. This region also contains a sequence that is repeated in the Chry5 genome outside the Ti plasmid, and that is widely distributed in the Rhizobiaceae.     

Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning

We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the lambda EXLX(+) and lambda EXLX(-) vectors that can be used for the expression in Escherichia coli of proteins encoded by cDNA inserts. This is achieved by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the lambda SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage lambda to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage lambda arms, lambda LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of a specialized plasmid between these sites will convert it into a phage lambda cDNA cloning vector with automatic plasmid subcloning capability.     

pUC12-STOP: An expression vector with portable translation stop signals

Summary A DNA linker with TAA translational stop codons in all three reading frames was inserted into the polylinker region of pUC12. The new plasmid pUC12-STOP is useful for the expression of DNA in cases where defined translational stops are desired. The STOP linker is flanked by unique restriction sites and thus can be excised as portable STOP linker fragments. The STOP linker was used to express in Escherichia coli a truncated form of the Herpes simplex virus type 1 glycoprotein D antigen.     

Plasmids pMB123 and pMB124--vectors for obtaining subfragments of DNA with random "sticky" ends

For preparing a DNA fragment with unique protruding ends, plasmid vectors pMB123 and pMB124 were constructed by inserting a synthetic polylinker into plasmid pUR222 at the EcoRI-PstI sites. The polylinker contains two FokI and HgaI sites at its ends in opposite orientation flanking a combination of SalGI, AccI, HindII, HindIII (the latter site is absent from pMB124) and BamHI sites. DNA fragment cloned at the SalGI and BamHI sites can be regenerated by either FokI or HgaI treatment, the SalGI and BamHI sites being deleted from the cloned sequence. Fragments coding for parts of human interleukin-2 were cloned in these vectors.     

Construction of pFRT,a Convenient Drosophila Transformation Vector with Functional FRT Sites

P-element transformation vector pCaSpeR3 was modified to obtain pFRT. The new vector contains two tandem FRT sites flanked by several unique restriction sites and separated by a polylinker harboring five convenient restriction sites, and allows easy cloning of DNA fragments between or close to the FRT sites. FLP-mediated excision of DNA sequences cloned between the FRT sites was demonstrated in vivo. The vector was proposed for molecular genetic studies of the position effect variegation, structural and molecular organization of Drosophila polytene chromosomes, etc.     

Repair of uracil residues closely spaced on the opposite strands of plasmid DNA results in double-strand break and deletion formation

Summary The role of closely spaced lesions on both DNA strands in the induction of double-strand breaks and formation of deletions was studied. For this purpose a polylinker sequence flanked by 165 by direct repeats was inserted within the tet gene of pBR327. This plasmid was used to construct DNA containing one or two uracil residues which replaced cytosine residues in the Kpnl restriction site of the polylinker. Incubation of the plasmid DNA construct with Escherichia coli cell-free extracts showed that double-strand breaks occurred as a result of excision repair of the opposing uracil residues by uracil-DNA glycosylase (in extracts from ung ⁺ but not in extracts from ung ^– E. coli strains). Recombination of direct repeats, induced by double-strand breakage of plasmid DNA, can lead to the deletion of the polylinker and of one of the direct repeats, thus restoring the tet ⁺ gene function which can be detected by the appearance of tetracycline-resistant colonies of transformants. Transformation of E. coli cells with single or double uracil-containing DNAs demonstrated that DNA containing two closely spaced uracil residues was tenfold more effective in the induction of deletions than DNA containing only a single uracil residue. The frequency of deletions is increased tenfold in an ung ⁺ E. coli strain in comparison with an ung ^– strain, suggesting that deletions are induced by double-strand breakage of plasmid DNA which occurs in vivo as a result of the excision of opposing uracil residues.     

Phagemid VPCS vectors for priming, cloning and sequencing

A phagemid was adapted for use as the vector in the vector-primer-cloner-sequencer cloning system. The use of this new vector markedly expanded the utility of this technology for the construction of cDNA libraries. Technological advantages and new capabilities include: (1) a greater number of unique restriction sites within the polylinker region; (2) the ability to produce single-stranded templates for nucleotide sequencing, and (3) a convenient means to synthesize strand-specific hybridization probes. With the use of this cloning system, a rat liver cDNA library (8.56 x 10(5) recombinants from 1 microgram of poly(A)+ RNA) was rapidly (in two days) constructed.