Genomic integration system based on pBR322 sequences for the cyanobacterium Synechococcus sp. PCC7942: transfer of genes encoding plastocyanin and ferredoxin.

Synechococcus sp. PCC7942 recipient strains were constructed for the chromosomal integration of DNA fragments cloned in any pBR322-derived vector, which carries the ampicillin resistance (ApR) marker. The construction was based on the incorporation of specific recombination targets, the so-called 'integration platforms', into the chromosomal metF gene. These platforms consist of an incomplete bla gene (ApS) and the pBR322 ori separated from each other by a gene encoding an antibiotic (streptomycin or kanamycin) resistance (SmR or KmR). Recombination between a pBR322-derived donor plasmid and such a chromosomal platform results with high frequency in restoration of the bla gene and replacement of the chromosomal marker (SmR or KmR) by the insert of the donor plasmid. The integration into the platform depends on recombination between pBR322 ori and bla sequences only and is therefore independent of the DNA insert to be transferred. The desired recombinants are found by selection for a functional bla gene (ApR) and subsequent screening for absence of the chromosomal antibiotic marker. Gene transfer with this integration system was found to occur efficiently and reliably. Furthermore, the presence of the pBR322 ori in the platform allowed for 'plasmid rescue' of integrated sequences. The system was applied successfully for the transfer of the gene encoding plastocyanin (petE1) from Anabaena sp. PCC7937 and for the integration of an extra copy of the gene encoding ferredoxin I (petF1) from Synechococcus sp. PCC7942 itself.     

Cloning and restriction mapping of the alkaline phosphatase structural gene (phoA) of Escherichia coli and generation of deletion mutants in vitro

The structural gene for alkaline phosphatase (phoA) of Escherichia coli was cloned into the PstI site of pBR322, from a transducing bacteriophage, lambda p(phoA-proC). The restriction map of the plasmid was established. Based upon this information, several phoA deletion plasmids as well as a smaller phoA+ plasmid were constructed. The genetic map and restriction map were correlated by recombination analysis. Cells carrying one of the phoA+ plasmids overproduce alkaline phosphatase 10-fold upon phosphate limitation. However, both regulation and processing of the enzyme were found to be normal.     

Highly efficient yeast-based in vivo DNA cloning of multiple DNA fragments and the simultaneous construction of yeast/ Escherichia coli shuttle vectors

In vivo recombinational cloning in yeast is a very efficient method. Until now, this method has been limited to experiments with yeast vectors because most animal, insect, and bacterial vectors lack yeast replication origins. We developed a new system to apply yeast-based in vivo cloning to vectors lacking yeast replication origins. Many cloning vectors are derived from the plasmid pBR322 and have a similar backbone that contains the ampicillin resistance gene and pBR322-derived replication origin for Escherichia coli. We constructed a helper plasmid pSUO that allows the in vivo conversion of a pBR322-derived vector to a yeast/E. coli shuttle vector through the use of this backbone sequence. The DNA fragment to be cloned is PCR-amplified with the addition of 40 bp of homology to a pBR322-derived vector. Cotransformation of linearized pSU0, the pBR322-derived vector, and a PCR-amplified DNA fragment, results in the conversion of the pBR322-derived vector into a yeast/E. coli shuttle vector carrying the DNA fragment of interest. Furthermore, this method is applicable to multifragment cloning, which is useful for the creation of fusion genes. Our method provides an alternative to traditional cloning methods.     

Direct cloning of cDNA inserts from lambda gt11 phage DNA into a plasmid vector by a novel and simple method

Bacteriophage lambda gt11 has been used quite extensively for producing cDNA libraries. The cDNA inserts are usually subcloned into a plasmid vector for large scale production and analysis. However, isolating the recombinant DNA of interest from the phage clones can be a tedious task. Since the E. coli strain Y1088 used for lambda gt11 phage infection carries a pBR322-derived plasmid endogenously, we reasoned that this endogenous plasmid could be used directly for cloning the cDNA phage insert. In this report, we describe a method in which cDNA inserts from lambda gt11 phage were cloned directly into the pBR322 plasmid vector, bypassing the time-consuming procedures of preparing plasmid DNA as a subcloning vector. This method is likely to be extended to the cloning of DNA inserts derived from other phage lambda vectors when bacteria containing endogenous pBR322 are used as host cells.     

Direct cloning of cDNA inserts from λgt11 phage DNA into a plasmid vector by a novel and simple method

Bacteriophage λgt11 has been used quite extensively for producing cDNA libraries. The cDNA inserts are usually subcloned into a plasmid vector for large scale production and analysis. However, isolating the recombinant DNA of interest from the phage clones can be a tedious task. Since the E. coli strain Y1088 used for λgt11 phage infection carries a pBR322-derived plasmid endogenously, we reasoned that this endogenous plasmid could be used directly for cloning the cDNA phage insert. In this report, we describe a method in which cDNA inserts from λgt11 phage were cloned directly into the pBR322 plasmid vector, by-passing the time-consuming procedures of preparing plasmid DNA as a subcloning vector. This method is likely to be extended to the cloning of DNA inserts derived from other phage λ vectors when bacteria containing endogenous pBR322 are used as host cells.     

Development of cloning vehicles from the Streptomyces plasmid pFJ103

A 20-kb plasmid, pFJ103, was isolated from a strain of Streptomyces granuloruber. A restriction endonuclease map of the plasmid was constructed. A Streptomyces gene that specifies resistance to the antibiotic thiostrepton was subcloned into Escherichia coli plasmid pBR322, inserted into pFJ103 and transformed into Streptomyces ambofaciens protoplasts. Two classes of transformants were obtained. One carries the pFJ104 plasmid consisting of the entire pFJ103 with the 1.8-kb thiostrepton resistance gene insert. The other carries the pFJ105 plasmid consisting of the 2.9-kb replicon segment of pFJ103 with the same thiostrepton resistance insert. A gene for neomycin resistance together with the entire E. coli pBR322 plasmid were cloned into pFJ105. The resulting E. coli-Streptomyces bifunctional vector, pFJ123, transformed both E. coli and Streptomyces. The small size of pFJ105, its ease of isolation, and efficient transformation of Streptomyces protoplasts establishes it, and its derivatives, as useful plasmid cloning vehicles for fundamental and applied studies     

人类泡沫逆转录病毒

根据 pHSRV1 3全基因组图谱和 pBR3 2 2质粒载体上限制酶切位点的性质 ,利用分子克隆技术 ,使 pHSRV1 3质粒上的bel1基因缺失突变并重新构建质粒载体 pHSRV1 3 -BR3 2 2 ,最后克隆到 1株含有目的基因组片段的重组质粒。     

Analysis of regulation of phoB expression using a phoB-cat fusion.

The phoB gene, which encodes a positive control factor for a number of phosphate-regulated genes in Escherichia coli, was cloned into multicopy plasmid pBR322. A phoB-cat fusion that expressed chloramphenicol transacetylase from the phoB promoter was constructed. Studies of the expression of the phoB-cat fusion showed that the pattern of regulation of the phoB gene was similar to that of the phoA gene, the structural gene for alkaline phosphatase. The phoB gene was derepressed under conditions of phosphate starvation, was constitutively expressed in a phoR background, and required the phoM gene product for expression in a phoR strain. Finally, a functional phoB product was required for its own synthesis. Our results indicate either that phoA gene expression responds directly to the concentration of the phoB gene product in cells or that the phoA and phoB controlling elements are quite similar.     

Transfer and maintenance of small, mobilizable plasmids with ColE1 replication origins in Legionella pneumophila

With the mutagenesis of specific, virulence-associated genes of Legionella pneumophila as the eventual goal, methods for gene transfer to these bacteria were developed. Following the observations of others that conjugative, broad-host-range plasmids could be transferred from Escherichia coli to L. pneumophila at low frequency, we constructed a small mobilizable vector, pTLP1, which carries oriV from pBR322, oriT from pRK2, Kmr from Tn5, and an L. pneumophila-derived fragment to permit chromosomal integration. In triparental matings including an E. coli with a conjugative (Tra+) helper plasmid, kanamycin-resistance was transferred from E. coli to L. pneumophila. Southern hybridization of L. pneumophila transconjugants showed that pTLP1 was replicated autonomously. Additional matings of plasmids having deletions or substitutions of pTLP1 sequences confirmed that replication in L. pneumophila requires oriV only. pTLP1 was maintained in L. pneumophila with passage on medium containing kanamycin but was rapidly lost after passage on nonselective medium. This plasmid instability in L. pneumophila is most likely due to rapid generation of plasmid-free segregants because of plasmid multimerization and low plasmid copy number. We conclude that mobilizable pBR322-derived plasmids can be used as shuttle vectors to transfer cloned genes to L. pneumophila, a feature that can be exploited for the purposes of mutagenesis or genetic complementation.     

HBV X 区基因部分序列的亚克隆与PCR 检测

目的：探讨HBVX区基因用于乙型病毒性肝炎早期诊断价值和意义。方法：设计特异性引物,将pBR322-HBV质粒X区部分序列PCR产物AT亚克隆至pBS—T载体,提取和纯化质粒DNA,再对HBVX区序列进行PCR扩增。结果：获得了预期希望的质粒。PCR的最佳退火温度为51℃,灵敏度达到101拷贝／2μl,线性范围101—1010拷贝／2μl。讨论：pBR322-HBV质粒中的靶基因成功地被亚克隆至pBS—T载体。pBR322-HBV中X区目的序列扩增产物为57bp,该小片段勿需纯化就可直接AT亚克隆至新载体,有利于后续的常规PCR检测和TaqMan MGB荧光定量PCR检测。     

大肠杆菌丝氨酸羟甲基转移酶基因(glyA)的克隆和表达

利用插入失活及营养缺陷型互补法将大肠杆菌K12 13kb的glyA基因克隆到质粒pBR329中。将重组质粒酶切,亚克隆,确定2.6kb PstI-EcoRI亚克隆片段带有完整的glyA基因。共获得12株glyA基因重组菌,对重组质粒进行了酶切鉴定。不同重组菌丝氨酸羟甲基转移酶(SHMT)活性及其酶表达量均不相同。受体菌未检测到丝氨酸的产生。重组菌株JM109(pSM13)、K12(pSM13)、K12(pSM14)和K12(pSM15)SHMT酶表达量分别占全菌可溶性蛋白的15.7％、15.4％、11.8％和9.48％。     

Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity

A Ti plasmid mutant was constructed in which all the on-cogenic functions of the T-DNA have been deleted and replaced by pBR322. This Ti plasmid, pGV3850, still mediates efficient transfer and stabilization of its truncated T-DNA into infected plant cells. Moreover, integration and expression of this minimal T-DNA in plant cells does not interfere with normal plant cell differentiation. A DNA fragment cloned in a pBR vector can be inserted in the pGV3850 T-region upon a single recombination event through the pBR322 region of pGV3850 producing a co-integrate useful for the transformation of plant cells. Based upon these properties, pGV3850 is proposed as an extremely versatile vector for the introduction of any DNA of interest into plant cells.     

Cloning, restriction endonuclease mapping and post-transcriptional regulation of rpsA, the structural gene for ribosomal protein S1

Transducing lambda phages have been isolated that carry segments of the Escherichia coli chromosome in the aspC region, 20.5 min on the E. coli map. One of these phages, lambda aspC2, carries rpsA, the structural gene for the ribosomal protein S1. A three kilobase fragment from this phage, cloned into either the plasmid pACYC184 or the plasmid pBR322, was found to express S1. In cells carrying the rpsA gene on the high copy number plasmid pBR322 the rate of rpsA mRNA synthesis was increased 40-fold, whereas the rate of protein S1 synthesis was doubled, in comparison with these rates in an rpsA haploid.     

Introduction of single-copy sequences into the chromosome of Escherichia coli: application to gene and operon fusions.

We have developed a general method for the introduction of any cloned sequence into the chromosome of Escherichia coli. This method employs an Hfr strain which carries a fragment of bla (the pBR322 gene imparting ampicillin resistance) between lacI and lacZ. Plasmid-borne inserts which are flanked by sequences from bla and lacZ can be introduced at this locus by homologous recombination. The isolation of recombinants is enhanced by selection for transfer of an integrated copy of the plasmid during conjugation. Once introduced into the chromosome, the inserted sequences can be transferred to other strains by conventional methods such as P1 transduction or conjugation. This method is suitable for the transfer of any cloned sequence to the chromosome and is particularly well suited to the construction of chromosomal gene and operon fusions with lacZ.     

Cloning of the Streptococcus thermophilus beta-galactosidase gene and its expression in Escherichia coli and Bacillus subtilis cells

The beta-galactosidase gene from the chromosome of Streptococcus thermophilus, strain 6 kb, has been cloned on a vector plasmid pBR322. The corresponding gene has been found to be located on the Pst1 DNA fragment. The restriction map of this 6 kb fragment has been constructed. The shortening of the DNA fragment carrying the beta-galactosidase gene has been achieved by digestion of the recombinant derivative of pBR322 by the restriction endonuclease Sau3A under the conditions of incomplete hydrolysis. The obtained fragments have been cloned into the BamHI site in the berepliconed shuttle vector pCB20 for grampositive and gramnegative bacteria. The obtained recombinant plasmids contained the beta-galactosidase gene in the inserted fragments of different length. Expression of the cloned beta-galactosidase gene in Escherichia coli and Bacillus subtilis cells has been studied.     

The plasmid cloning vector pBR325 contains a 482 base-pair-long inverted duplication

The plasmid pBR325 is a cloning vector constructed in vitro by addition of the chloramphenicol resistance (Cmr) gene of an IS1-flanked transposon to pBR322 (Bolivar, 1978). It is a 5 995 bp plasmid carrying no sequence originating from IS1. DNA-sequence data suggest that its Cmr segment was derived from a Cm transposon longer than Tn9. The plasmid pBR325 carries between the Cmr and Tcr genes a 482 bp sequence which duplicates, in the opposite orientation, a section pf pBR322 located at the end of the tcr gene. The same structure was found in pBR328, a deletion derivative of pBR325 (Soberon et al., 1980). The possible implications of this inverted duplication on cloning experiments are discussed.     

Selection of lambda Spi- transducing phages using the P2 old gene cloned onto a plasmid

The old gene product of the P2 prophage interferes with plaque formation by lambda wild type phage but allows lambda phages whose red and gam genes have been deleted to form small, visible plaques (the lambda Spi- phenotype). The old gene product also kills Escherichia coli recB or recC mutants. We have cloned the old gene into the high-copy-number plasmid pBR322, where it prevents plaque formation by both lambda Spi+ and lambda Spi- phages. We transferred a DNA fragment that carries the old gene to the low-copy-number plasmid pSC101 and found that lambda Spi- phages can be selected on strains that carry this plasmid. The plasmid-borne old gene kills E. coli recB mutants, providing a selection for old- mutants.     

Fate of cloned bacteriophage T4 DNA after phage T4 infection of clone-bearing cells

Plasmid pBR322 replication is inhibited after bacteriophage T4 infection. If no T4 DNA had been cloned into this plasmid vector, the kinetics of inhibition are similar to those observed for the inhibition of Escherichia coli chromosomal DNA. However, if T4 DNA has been cloned into pBR322, plasmid DNA synthesis is initially inhibited but then resumes approximately at the time that phage DNA replication begins. The T4 insert-dependent synthesis of pBR322 DNA is not observed if the infecting phage are deleted for the T4 DNA cloned in the plasmid. Thus, this T4 homology-dependent synthesis of plasmid DNA probably reflects recombination between plasmids and infecting phage genomes. However, this recombination-dependent synthesis of pBR322 DNA does not require the T4 gene 46 product, which is essential for T4 generalized recombination. The effect of T4 infection on the degradation of plasmid DNA is also examined. Plasmid DNA degradation, like E. coli chromosomal DNA degradation, occurs in wild-type and denB mutant infections. However, neither plasmid or chromosomal degradation can be detected in denA mutant infections by the method of DNA--DNA hybridization on nitrocellulose filters.     

Promoter-probe vectors for the analysis of divergently arranged promoters

A series of plasmid-based promoter-probe vectors has been constructed which are particularly useful for the analysis of divergent control regions. Each vector contains a pair of divergently oriented indicator genes whose expression can be monitored over a wide range by simple assay methods. These genes are separated by different polylinkers. Specifically, the beta-galactosidase gene (lacZ) was employed in combination with either the galactokinase gene (galK) or the alkaline phosphatase gene (phoA). In all cases translational stop codons are present in all three reading frames upstream from the initiation codon. The vectors permit direct detection of promoters--independent of insert orientation--on indicator plates after transformation. Using this vector system, we further characterized the divergent tet control regions of transposon Tn10 and plasmid pBR322.