Expression and stability of a recombinant plasmid in Zymomonas mobilis and Escherichia coli

A recombinant plasmid was constructed by ligating EcoRI digests of the plasmid cloning vector pBR325 and pZMO2, one of the natural plasmids of Zymomonas mobilis ATCC 10988. This vector, named pDS212 (total size 7.9 kb), which was able to transform Escherichia coli efficiently, was also transferred to Z. mobilis hosts by mobilization during conjugation using the helper plasmid pRK2013. pDS212 was inherited stably in both E. coli and Z. mobilis hosts and could be recovered intact from them. Markers of pBR325 and pRK2013 were also transferred in Z. mobilis but at very low frequencies. Neither pBR325 nor pRK2013 could be recovered intact from the Z. mobilis hosts. It is proposed that expression and stability of pDS212 in Z. mobilis is due to the origin of replication of pZMO2 that it carries, and that it may be used for developing a gene transfer system in Z. mobilis.     

Molecular cloning of a Bacillus subtilis xylanase gene in Escherichia coli

A gene coding for xylanase synthesis in Bacillus subtilis was isolated by direct shotgun cloning using Escherichia coli as a host. Following partial digestion of B. subtilis chromosomal DNA with PstI or EcoRI restriction enzymes, fragments ranging from 3 to 7 kb were introduced into the PstI or EcoRI sites of pBR325. Transformed colonies having lost either the ampicillin or chloramphenicol resistance markers were screened directly on 1% xylan plates. Out of 8000 transformants, ten xylanase-positive clones were identified by the clearing zone around lysozyme-treated colonies. Further characterization of one of the clones showed that the xylanase gene was present in a 3.9-kb insert within the PstI site of the plasmid pBR325. Retransformation of E. coli strain with the xylanase-positive hybrid plasmid pRH271 showed 100% transformation to xylanase production. The intracellular xylanase produced by the transformed E. coli was purified by ion exchange and gel permeation chromatography. The electrophoretic mobility of the purified xylanase indicated an Mr of 22 000.     

Identification of the Escherichia coli K-12 cpxA locus as a single gene: construction and analysis of biologically-active cpxA gene fusions

In the accompanying communication we showed that a 2 kb EcoRI-BamHI restriction fragment from the pfkA-rha interval of the Escherichia coli K-12 chromosome fully complemented a chromosomal cpxA mutation when the fragment was cloned in pBR325. The same fragment cloned in pBR322 lacked any complementing activity. We show here that minicells containing the pBR325 derivative (pRA310) synthesized a 33 kDa polypeptide, designated phi 33, that was not synthesized in minicells containing the pBR322 derivative (pRA311) or either of the parent plasmids. Synthesis of the phi 33 polypeptide did not occur in minicells containing Tn5 insertion alleles of pRA310 that inactivated its cpxA complementing activity. These insertions mapped within the vector cat (chloramphenicol acetyltransferase gene) sequence immediately adjacent to the EcoRI site of pRA310 and within the 700-800 bp of the cloned EcoRI-BamHI fragment immediately adjacent to the EcoRI site. Tn5 insertions located within the fragment but closer to the BamHI terminus affected neither the cpxA complementing activity of pRA310 nor synthesis of the phi 33 polypeptide in minicells. Plasmid pRA311 could be converted to a plasmid with cpxA complementing activity by cloning into its EcoRI site a restriction fragment containing a hybrid trp-lacUV5 promoter, the lacZ ribosome binding site, and the first eight lacZ codons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of deletion derivatives of an autonomously replicating Neurospora plasmid.

We previously described two plasmids that replicate autonomously in both Neurospora and E. coli (Stohl and Lambowitz, Proc. Natl. Acad. Sci., U.S.A. 80, 1058-1062, 1983). One plasmid, pALS1, consists of the Neurospora ga-2+ gene (3 kb Hind III-fragment), the mitochondrial plasmid from N. intermedia strain P405-Labelle, and E. coli plasmid pBR325. The other, pALS2, is a putative deletion derivative of pALS1 that lacks most or all of the Labelle insert and that was repeatedly recovered from Neurospora transformants. We have now sequenced the region encompassing the deletion in five pALS2 plasmids isolated independently in two different laboratories. All five plasmids are identical in this region and completely lack the Labelle insert. We have also characterized an additional deletion derivative that retains a small (approximately 0.5 kb) segment of the Labelle insert. The results for pALS2 suggest that pBR325 plus the ga-2+ segment constitute a Neurospora replicon.     

Mapping and cloning of the gene coding for beta-glucanase from various bacilli

Beta-glucanase gene from Bacillus subtilis 168 has been mapped by bacteriophage pBS1 transduction technique between sacA and purA genes. The stimulating effect of pleiotropic mutations pap, amyB and sacUh on beta-glucanase production in Bacillus subtilis and Bacillus amyloliquefaciens has been described. Beta-glucanase gene from Bacillus amyloliquefaciens has been cloned ona Charon 4A vector. Expression of the gene in E. coli cells depended on the orientation of the cloned DNA on a pBR322 vector plasmid. Maximal enzymatic activity was registered in periplasm. Beta-glucanase gene was recloned in Bacillus subtilis cells. Bacillus subtilis strain, harbouring pBG1, produces 500 times more beta-glucanase as compared with the wild type strain of Bacillus subtilis.     

Increased stability of pBR322-related plasmids in Escherichia coli W3101 grown in carrageenan gel beads

Abstract The stability and the copy number of pBR322, pBR325 and pBR328 were studied during continous cultures of free and immobilized E. coli W3101 without selective pressure. In the free-cell system, it was found that pBR328 and pBR325-free E. coli cells appeared after a lag period. They rapidly overgrew the cultures and the plasmid copy number subsequently declined. On the other hand, an increase in the proportion of pBR322- carrying cells during a free continuous culture was observed. This increase correlated with that of plasmid copy number. By contrast, in the immobilized- cell system, plasmid free segregants were not detected in all the cases even after 250 generations. We have also shown that plasmid copy number remained constant and phenomena such as fluctuations or genetic modifications which occured after long term growth of bacteria in a free continuous culture could be avoided throughout cell immobilization.     

Expression of a Thiobacillus ferrooxidans origin of replication in Escherichia coli.

A cryptic plasmid from an autotrophically grown arsenic-resistant strain of Thiobacillus ferrooxidans was isolated and cloned into pBR325. The origin of replication of pBR325 was deleted, and the recombinant plasmid was shown to replicate in Escherichia coli, using an origin of replication located on the Thiobacillus plasmid.     

Cloning and delimiting one chloroplast DNA replicative origin of Chlamydomonas.

The EcoR1 restriction fragments containing D-loops which marked the replication origin of chloroplast DNA were identified in two different species of Chlamydomonas. Each fragment was cloned in the E. coli plasmid pBR325. The cloned fragments were compared by restriction endonuclease analyses and by heteroduplex analyses in the electron microscope. The relative position of the D-loop regions and the homologous regions between the 2 fragments was determined. The D-loops were located within one short homologous region of 0. 42kb in length between the 2 cloned restriction fragments. The homologous region was subcloned in pBR322. Closed circular plasmid DNAs containing the short homologous region showed preferred denaturation in the D-loop region under physiological salt concentration which suggested that D-loop region was AT rich. Sequence divergence was detected at both ends of the D-loop region. Southern blot analyses indicated the presence of species-specific repetitive sequences within the divergent regions.     

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％。     

Persistence of pBR322-related plasmids in Escherichia coli grown in chemostat cultures

Abstract The stability of plasmid pBR322 and a number of close derivatives was examined by continuous culture of Escherichia coli . Cultures were subjected to either glucose, phosphate or magnesium limitation in non-selective medium at a dilution rate of 0.1/h. Under these conditions pBR322 was eventually lost from the population, but only after a distinct lag period. The closely related plasmids pBR325 and especially pBR327 and pBR328, but not pAT153, were lost more rapidly. Three cosmids pHC79, pSJ55 and pJB8 were generally found to be less stable than the pBR322-type plasmids from which they were derived. Chimaeric plasmids containing DNA from yeast and from a thermophilic bacillus were also unstable in E. coli .     

Location and molecular cloning of the structural gene for the deoxyguanosine triphosphate triphosphohydrolase of Escherichia coli

The structural gene for deoxyguanosine triphosphate triphosphohydrolase (dGTPase) (EC 3.1.5.1) and its regulator, optA, have been located on a lambda phage carrying a 17.5kb Escherichia coli DNA insert. The DNA fragment has been excised and ligated into pBR325 and also transferred to another lambda vector. From the results of transduction and transformation experiments, we find that the structural gene for dGTPase is very closely linked to optA and dapD, which locates it at approximately 3.6 minutes on the genetic map of E. coli K12. We propose the mnemonic dgt as the designation for the structural gene for this enzyme.     

Cloning of genes encoding pectolytic enzymes from a genomic library of the phytopathogenic bacterium, Erwinia chrysanthemi

Erwinia chrysanthemi are phytopathogenic enterobacteria causing soft-rot disease due to pectolytic enzymes degrading plant cell walls. We constructed a genomic library from Sau3A-digested E. chrysanthemi B374 DNA cloned in the BamHI site of the broad-host-range cosmid pMMB33 grown in Escherichia coli. Out of 1500 kanamycin-resistant (KmR) transductants of E. coli, nine pectolytic-enzyme-positive clones were identified. One of these contained the pEW325 cosmid with a 35-kb insert of Erwinia DNA. Cell extracts of E. coli harboring the cosmid pEW325 were fractionated on a polyacrylamide electrofocusing gel; bands with pectolytic activity were found to co-focus with pectolytic enzymes of E. chrysanthemi B374 strain. Cosmid pEW325 encodes three pectolytic enzymes PL10, PL20 and PL130 with isoelectric points of about 9.3, 9.2 and 4.6, respectively. These enzymes are lyases that cleave polygalacturonate by transelimination, and give rise to unsaturated products. A 15-kb HindIII fragment coding for polygalacturonate lyases was subcloned in pBR322, and a physical map of the resulting plasmid pPL01 was constructed. Starting from the pPL01, various endonuclease-generated fragments were subcloned into pBR322. Genes encoding pectate lyases were localized within an 8-kb fragment (pPL04) and then in a 2.7-kb fragment (pPL03). Polygalacturonate lyases are expressed at various levels; they accumulated in the periplasmic space of E. coli host, whereas E. chrysanthemi secreted these enzymes into the culture medium.     

Cloning the uridine phosphorylase (udp) gene of Escherichia coli and its expression in recombinant plasmids

On the basis of Escherichia coli DNA and vectors pBR322, pUC19, hybrid plasmids restoring Udp+ phenotype in the E. coli deletion (delta udp) mutant have been obtained. The udp gene is carried by a 8 kb PstI fragment (on the pUD2) and by a smaller 2.87 kb PstI-SalGI fragment from the PstI fragment (pUD7). The uridine phosphorylase level was 30 times higher in the cells containing hybrid plasmid as compared to the strain with chromosomal location of the udp gene. On the other hand, the measurements of uridine phosphorylase activity in the cytR- and cya- background indicate that expression of the cloned udp gene escapes partially negative control of the CytR repressor and positive control of cAMP--CRP complex. These data suggest that the 2.87 kb PstI--SalGI-fragment contains the intact udp gene which is transcribed from its own promoter. Increase in the activity of beta-galactosidase encoded by udp-lacZ fusion has been observed in the presence of pUD2 or pUD7, which was suggested to be the consequence of titration of CytR repressor molecules in the operator region of the cloned udp.     

Molecular analysis of an aesculinase activity from Klebsiella oxytoca

A DNA fragment containing a Klebsiella oxytoca gene for aesculinase activity was cloned on the multicopy plasmid pBR322. This β-glucosidase activity was confined to aesculin hydrolysis only. It was expressed equally well in Escherichia coli, Salmonella typhimurium and Aeromonas hydrophila. Two polypeptides were found to be encoded within the 2·6 kb of the cloned DNA encoding aesculinase activity.     

Structural and functional organization of the transposon Tn2555 carrying saccharose utilization genes

Transposon Tn2555 was isolated from a clinical E. coli strain carries the genes for sucrose utilization. Previously it was shown that Tn2555 is very unstable and undergoes structural rearrangements with a high frequency. Several deletion derivatives of Tn2555 and one with an inversion of the internal segment were found. They form the Tn2555 transposon family. This paper describes further structural and functional analysis of Tn2555. In the course of the experiments on pBR325 (Mob-) mobilization by conjugative RP4 derivatives, containing Tn2555 family elements, it was found, that all of them induce cointegrate formation. Some of these cointegrates were able to dissociate in rec+ and recA E. coli cells. Restriction endonuclease analysis of the resulting plasmids have shown, that among them were the end products of the Tn2555 transposition from RP4 to pBR325. Besides, the pBR325 derivatives, containing a discrete DNA segment of approximately 800 b.p., originating from Tn2555, were found. The segment can transpose from pBR325 to RP4 indicating that it is an insertion sequence. This new IS-element was designated IS286. The size and the genetic properties of IS286 resemble those of the IS1 element. However restriction analysis and Southern hybridization data show no significant homology between IS286 and IS1. It was found that the Tn2555 family elements are flanked by directly repeated IS286. One of them (Tn2555.3) contains an additional copy of IS286 in its internal region.     

An additional restriction fragment length polymorphism at the thyroglobulin gene.

The study was aimed at the screening of human chromosomal DNA for restriction fragment length polymorphism (RFLP) at the human thyroglobulin (hTg) gene locus. The RFLP screening was performed in a typical way. As hybridization probes were used 5 Pst I fragments of hTg cDNA of the total length 5.1 kb pairs cloned in pBR 322. One not described polymorphism was found by using the probe hTg 10, (nucleotides from position 4830 to 5810 in the 3' flanking region of hTg). Restriction enzyme Msp I identified a single two allele polymorphism: A1: 3.5 kb and A2: 2.5 kb. Of 32 unrelated healthy individuals two were homozygous for 3.5 kb, one was homozygous 2.5 kb and 29 were heterozygous for both 3.5 kb. and 2.5 kb. Thus, the frequencies of the 3.5 and 2.5 kb Msp I alleles were 0.52 and 0.48 respectively.     

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.     

Genetic transformation of somatic cells. III. An analysis of the status of the plasmid nucleotide sequences in the extrachromosomal DNA of transformant clone cells and the rescue of extrachromosomal molecules of the plasmid DNA

Extrachromosomal DNAs from TK+ transformant clones of A238 Chinese hamster cells isolated after the treatment with plasmid pST826 containing thymidine kinase gene (TK-gene) of Herpes simplex virus (HSV1) and 1.8 kb insert of human satellite III DNA (HSIII) were studied by hybridization technique. In two TK+-clones (2T301 and 2T16) large quantities of rearranged plasmid DNA molecules were found. Electron microscopy show in clone 2T301 the presence of circular DNAs with average length being 4.64 +/- 0.27 kb. These molecules were rescued by retransformation into E. coli and analysed by restriction mapping and hybridization. All of them contain deletions spanning the entire TK gene of HSV1 and pBR325 sequences situated just downstream from the ORI of replication. The origin of extra-replicating circular DNA in 2T301 clone is discussed.     

Cellulase genes of Cellulomonas uda CB4. II. Cloning and expression of a CM-cellulose hydrolyzing enzyme (endoglucanase) gene in Escherichia coli

A CM-cellulose hydrolyzing enzyme (endoglucanase, CMCase) gene in Cellulomonas uda CB4 was cloned in E. coli with pAT325 constructed from pAT153 and pBR325. Plasmid pCM41 was isolated from the transformant producing CMCase, and the CMCase gene cloned was a 4.8 kb BamHI fragment. About 70% of the CMCase activity was observed in the periplasmic space of E. coli carrying pCM41. The optimal pH and temperature for the CMCase thus cloned were pH 6–8 and 55–60°C, respectively, as was the case for the donor.     

Amplification of the adenylate cyclase gene in Escherichia coli cells

Amplification of the cya gene of E. coli on the plasmid pBR325 leads to an increase of adenylate cyclase activity proportional to the gene dosage. In strains harboring hybrid plasmids with cya gene the intracellular level of cAMP and the rate of nucleotide secretion are also elevated. The adenylate cyclase activity in cells with truncated cya gene cloned on pBR322 remains sensitive to glucose inhibition. Amplification of the cya gene leads to considerable resistance of beta-galactosidase synthesis to transient repression by alpha-methylglucoside, but does not influence the permanent repression caused by glucose.