Plasmid Vector pUC19 Research Articles

Cloning of Mycobacterium smegmatis Exochelin MS genes fxbA, fxbC and exiT in Escherichia coli

Siderophores are low molecular weight compounds with a strong affinity for iron. For instance, exochelin MS is a siderophore produced by M. smegmatis. In the present study, we have cloned exochelin MS biosynthesis genes of Mycobacterium smegmatis, namely, fxbA, fxbC and exiT in E. coli DH5α. These genes were amplified by PCR and cloned in plasmid vector pUC19. The clones harbouring the recombinant plasmids were isolated, purified and sequenced. The sequencing and the BLAST results using NCBI database confirmed the success of cloning fxbA, fxbC and exiT genes.

Enzymes for the laundry industries: tapping the vast metagenomic pool of alkaline proteases

SummaryIn the wide field of laundry and cleaning applications, there is an unbroken need for novel detergent proteases excelling in high stability and activity and a suitable substrate range. We demonstrated the large amount of highly diverse subtilase sequences present in metagenomic DNA by recovering 57 non‐redundant subtilase sequence tags with degenerate primers. Furthermore, an activity‐ as well as a sequence homology‐based screening of metagenomic DNA libraries was carried out, using alkaline soil and habitat enrichments as a source of DNA. In this way, 18 diverse full‐length protease genes were recovered, sharing only 37–85% of their amino acid residues with already known protease genes. Active clones were biochemically characterized and subjected to a laundry application assay, leading to the identification of three promising detergent proteases. According to sequence similarity, two proteases (HP53 and HP70) can be classified as subtilases, while the third enzyme (HP23) belongs to chymotrypsin‐like S1 serine proteases, a class of enzymes that has not yet been described for the use in laundry and cleaning applications.

Two novel gastric cancer-associated genes identified by differential display.

AIM:To clone novel gastric cancer-associated genes and investigate their roles in gastric cancer occurrence.METHODS:A method called differential display was used which allows the identification of differentially expressed genes by using PAGE to display PCR-amplified cDNA fragments between gastric cancer cells and normal gastric mucosa cells. These fragments were cloned into plasmid vector pUC18. Homology analysis was made after sequencing these fragments.RESULTS:Two novel genes were identified compared with sequences from GenBank. One was registered with the AD number AF 051783.In situ hybridization showed that these two novel genes expressed specifically in gastric cancer tissues.CONCLUSION:The two novel genes obtained by differential display were confirmed to be gastric cancer-associated genes using in situ hybridization.

Sequencing and expression analysis of a Schistosoma mansoni gene homologue to a Drosophila gene involved in germ plasm assembly.

Genes coding for proteins involved in generegulation and/or development are of great inter-est in the study of the biology of Schistosomamansoni. This trematode is the etiologic agent ofschistosomiasis and presents a complex life cyclewith drastic morphologic changes between stages.Recently, some strains have become resistant tothe drugs currently in use to eradicate the disease(D Cioli et al. 1995 Pharmac Ther 68: 35-85). Thestrategy of gene discovery program in S. mansoniby using the EST (expressed sequence tag) ap-proach (GR Franco et al. 1995 Gene 152 : 141-147)has been very efficient in the discovery of new S.mansoni genes, which were unlikely to be identi-fied using classical procedures based on pheno-type. A class of genes that interested us particu-larly were those that in other organisms wereknown to be involved in the regulation of embryo-genesis. Among these, we selected one that pre-sented a high homology to a Drosophila genenamed mago nashi. In diptera this gene is involvedin the process of germ plasm assembly and itsmutation results in sterility of F1 progeny and alsoin the formation of the perpendicular axes (REBoswell et al. 1991 Development 113: 373-384).We reasoned that this gene might conceivably playa role in the morphogenetic changes seen in thelife cycle of the parasite . We thus decided to char-acterize S. mansoni mago nashi further by obtain-ing its full length cDNA and genomic sequences,as well as studying its expression pattern at thedifferent life stages of the worm.The whole cDNA was sequenced in both di-rections yielding 485 nucleotides (nt) that codedfor a protein of 146 amino acids with 84% of ho-mology to the Drosophila homologue (Fig. 1). Pro-cedures for plasmidial DNA preparation, sequenc-ing and analysis of sequences have been previouslydescribed (Franco loc. cit.). From the cDNA se-quence, primers were designed and the genomicgene amplified from S. mansoni total DNA. Thegenomic amplified product was bigger than thecorrespondent cDNA amplified product when twodifferent pairs of primers were used (Fig. 2). ThePCR amplification products were cloned in a plas-mid vector (pUC18) by using the Pharmacia Sureclone kit and sequenced in both directions usingfluorescent primers in an automated DNA se-quencer. Consistent with the results of Fig. 2, threeintrons were identified in the genomic sequence,two of 34 nt and one with 33 nt producing a totalof a 101 nt of intron sequences (Fig. 3).Our next step was to investigate the gene ex-pression pattern at the different life cycle stages.Through the RT-PCR technique using cDNA ob-tained from different stages and specific primers,it was shown that mago nashi is expressed in allstages studied i.e. egg, schistosomula and adultworm (data not shown). This was not unexpectedalthough this gene was first identified in droso-phila embryos, it was also identified in adult flies(PA Newmark & RE Boswell 1994 Development120: 1303-1313). The gene had also been shownto be expressed in a large variety of human adulttissues such as lung, kidney, liver, heart, pancreas,brain and placenta (X- Zhao et al. 1998 Genomics47: 319-322).Since this work was started, the mago nashigene has been identified in a variety of differentorganisms. These include mouse, human,Caenorhabditis elegans, Brugia malayi,Arabidopsis thaliana and Oryza sativa . Alignmentof the conceptual translations of these mago genes

Cloning, Sequencing, and Recombinant Expression ofNAT1, NAT2,andNAT3Derived from the C3H/HeJ (Rapid) and A/HeJ (Slow) Acetylator Inbred Mouse: Functional Characterization of the Activation and Deactivation of Aromatic Amine Carcinogens

An acetylator polymorphism has been described in the mouse and the inbred strains C3H/HeJ and A/HeJ constitute rapid and slow acetylators, respectively. TheNAT1, NAT2,andNAT3genes from C3H/HeJ and A/HeJ acetylator inbred mouse strains were amplified using the polymerase chain reaction, cloned into the plasmid vector pUC19, and sequenced. They were then subcloned into the prokaryotic expression vector pKK223-3 and expressed inEscherichia colistrain JM105. The 870-bp nucleotide coding region ofNAT1andNAT3did not differ between the rapid and slow acetylator mouse strains, or from that of previously published mouseNAT1andNAT3sequences. However,NAT2did differ between the rapid and slow acetylator strains with an A296T transition which causes a (Asn99→ Ile) substitution in the deduced amino acid sequence. Recombinant NAT1, NAT2, and NAT3 proteins catalyzedN-, O-,andN,O-acetyltransferase activities. NAT3 catalyzed aromatic amineN-acetyltransferase activities at very low rates, which confirms a previous study. ApparentKmandVmaxkinetic constants forN-acetylation were 5- to 10-fold lower for recombinant mouse NAT1 than NAT2. Intrinsic clearances for recombinant mouse NAT1- and NAT2-catalyzedN-acetylation of aromatic amine carcinogens were comparable. Both recombinant mouse NAT1 and NAT2 catalyzed the metabolic activation ofN-hydroxyarylamine (O-acetylation) andN-hydroxyarylamide (N,O-acetylation) carcinogens. Recombinant mouse NAT3 catalyzedN,O-acetylation at very low rates, whileO-acetylation was undetectable. No difference was observed between rapid and slow acetylator recombinant NAT2 proteins to activate aromatic amines byO- orN,O-acetylation, in substrate specificity, expression of immunoreactive protein, electrophoretic mobility, orN-acetyltransferase Michaelis–Menten kinetic constants. However, the slow acetylator recombinant NAT2 protein was over 10-fold less stable than rapid acetylator recombinant NAT2. These studies demonstrate metabolic activation and deactivation by recombinant mouse NAT1, NAT2, and NAT3 proteins and confirm and extend previous studies on the molecular basis for the acetylation polymorphism in the mouse.

Cloning and expression of an agarase gene from a marine bacterium Pseudomonas sp. w7

Two different agarase genes (pSW1, pSW3) were cloned from a marine bacterium Pseudomonas sp. W7 into E. coli JM83 using the multicopy plasmid vector pUC19. Two cloned strains of recombinant E. coli which showed the agarase activity were obtained and were named E. coli JM83/pSW1 and E. coli JM83/pSW3. These strains had the insert fragment of 3.7kb and 3.0kb, respectively. The N-terminal amino acid sequence of the agarase containing the recombinant plasmid pSW3 was determined and the sequence did not show homology to any other known agarases. The optimum pH and temperature of the agarases from the cloned strains, E. coli JM83/pSW1 and pSW3, were 6.0, 7.0 and 30°C, 40°C, respectively.

Construction of pRES18 and pRES19, Streptomyces-Escherichia coli shuttle vectors carrying multiple cloning sites

We developed two Streptomyces-Escherichia coli shuttle vectors. The plasmid pRES102, consisting of the essential region of pRES1 and the thiostrepton resistance gene ( tsr) fragment of pIJ702, was combined with the E. coli plasmid vector pUC18 or pUC19. The resulting shuttle vectors, designated pRES18 and pRES19, respectively, have relatively compact size (6.25 kb), low copy number, multiple cloning sites reciprocally arranged in opposite directions, and selection markers for both Streptomyces ( tsr) and E. coli (β-lactamase ( bla) and β-galactosidase ( lacZ)). These shuttle vectors are capable of carrying DNA fragments as long as 10 kb, of being maintained in S. griseus, S. lavendulae and S. lividans, and are compatible with pIJ702.

Polymerase chain reaction for detecting Escherichia coli O157:H7

Escherichia coli O157:H7 is known as an important cause of hemorrhagic colitis and hemolytic uremic syndrome. Real-time procedures that are sensitive for detecting small populations of this bacterium in food are lacking and needed. An expression library was constructed by ligation of BamHI- EcoRI DNA fragments of E. coli O157:H7 to plasmid vector pUC19 and transformation of recombinant plasmids to E. coli JM109. A clone that contained a specific DNA fragment of E. coli O157:H7 was identified by colony immunoblot assay using monoclonal antibody MAb 4E8C12 that uniquely links to E. coli O157:H7 and a few other serotypes of verotoxin-producing E. coli. The DNA sequence of the clone consisted of 110 bp of 5′ region of enterohemorrhagic E. coli (EHEC) eae gene and a 688 bp DNA fragment adjacent to 5′ end of the eae gene, including an unknown function gene encoding 156 amino acids. A pair of oligonucleotide primers was synthesized based on the sequence of the 688 bp fragment. The primers were used in a polymerase chain reaction (PCR) to amplify a target DNA of 633 bp. The primers amplified 1 ng of DNA from 67 strains of E. coli O157:H7, two strains of E. coli O157:NM, and 7 of 11 E. coli O55:H7 and O55:NM strains, but not 50 ng of DNA from 34 strains of 29 other E. coli serotypes and 25 strains of 8 other bacterial species. Annealing temperatures from 60 to 63 °C could be used for the PCR without loss of specificity. The minimum amount of target DNA detected by the PCR was 5 pg. When a boiling method and GeneReleaser were used, the PCR was able to detect as few as 25 and 38 CFU of E. coli O157:H7, respectively, in 3 h.

Nucleotide sequence of the SrRNA gene and phylogenetic analysis of Trichomonas tenax.

The small subunit ribosomal RNA (SrRNA) gene of Trichomonas tenax ATCC30207 was amplified by PCR and the 1.55-kb product was cloned into plasmid vector pUC18. Four clones were isolated and sequenced. The insert DNAs were 1,552 bp long and their G+C contents were 48.1%; three of them had exactly the same DNA sequences and one had only one nucleotide change. A representative SrRNA sequence was analyzed and a phylogenetic tree was estimated by the neighbor-joining (NJ) method. Among the protists examined, T. tenax was placed as the closest relative of Tritrichomonas foetus, as expected from the traditional taxonomy. The total homology between the two SrRNA sequences was 89.2%.

Nucleotide sequence of the SrRNA gene of Entamoeba gingivalis: applications for construction of a species-specific DNA probe and phylogenetic analysis.

The small subunit ribosomal RNA (SrRNA) gene of Entamoeba gingivalis was amplified by PCR and the product of 1.9-kbp sequence was cloned into a plasmid vector pUC18. Four clones were isolated and sequenced. The insert DNAs were 1918- to 1921-bp long and A+T rich (65.5%). The four SrRNA sequences of E. gingivalis were found to be aligned with those of nine related protozoans while searching for E. gingivalis-specific sequences. A sequence of 28 oligonucleotides was chosen, chemically synthesized, and labeled with digoxigenin for use as a DNA probe. The probe thus constructed was shown to hybridize only with either the SrRNA-coding DNAs or the cells of the two E. gingivalis strains and not with those of other protozons or oral fungi tested. A representative SrRNA-sequence was analyzed and a phylogenetic tree was constructed by the neighbor-joining (NJ) method. Among the protists examined, E. gingivalis was placed next to Entamoeba histolytica as expected from the traditional taxonomy.

Features of the DNA fingerprinting probe pITZ1

Stringently controlled plasmids generate DNA fingerprint patterns in mammals when used at low hybridization temperatures. In order to develop a probe for use in paternity testing in cattle we screened a bovine, partial genomic plasmid library with the PCR-amplified ori region of plasmid P1. Of eight isolated clones one generated strong band patterns at high stringency in various mammalian species (data not shown). Sequence analysis revealed an imperfect, compound dinucleotide repeat region, which was PCR-amplified and cloned into the plasmid vector pUC19. Fingerprint results generated by this probe (termed pITZ1) in cattle are compared with the results generated by VNTR-probe pV47, which itself was developed by screening a human chromosome 16 library with tandem repeats of bacteriophage M13. Probe pITZ1 is useful in conjunction with other VNTR-probes for DNA fingerprinting in cattle and donkey populations. The dinucleotide repeat region responsible for the band patterns generated with pITZ1 is close to an Alu-like sequence, which may be involved in eukaryotic replication mechanisms.

Confirmation that DNA encoding the major fimbrial subunit of Av24 fimbriae is homologous to DNA encoding the major fimbrial subunit of F107 fimbriae

Plasmid DNA from porcine enterotoxigenic Escherichia coli strain Av24 (O 141:K85ab) was cloned into recipient E. coli strain JM105 using the plasmid vector pUC18. Clones were obtained that produced fimbriae which reacted with antisera specific to the fimbriae produced by strain Av24. Restriction mapping of cloned DNA, PCR with fedA primers and DNA sequencing showed a portion of the cloned DNA to be homologous to that encoding the major fimbrial subunit of F107 fimbriae. This confirms that the fimbriae possessed by strains of E. coli causing both edema disease and post-weaning diarrhoea in piglets are variants of the same fimbriae.

Confirmation that DNA encoding the major fimbrial subunit of Av24 fimbriae is homologous to DNA encoding the major fimbrial subunit of F107 fimbriae

Plasmid DNA from porcine enterotoxigenic Escherichia coli strain Av24 (O141:K85ab) was cloned into recipient E. coli strain JM105 using the plasmid vector pUC18. Clones were obtained that produced fimbriae which reacted with antisera specific to the fimbriae produced by strain Av24. Restriction mapping of cloned DNA, PCR with fedA primers and DNA sequencing showed a portion of the cloned DNA to be homologous to that encoding the major fimbrial subunit of F107 fimbriae. This confirms that the fimbriae possessed by strains of E. coli causing both edema disease and post-weaning diarrhoea in piglets are variants of the same fimbriae.

Diversity of cultivable and uncultivable oral spirochetes from a patient with severe destructive periodontitis

To determine the genetic diversity of cultivable and uncultivable spirochetes in the gingival crevice of a patient with severe periodontitis, partial 16S rRNA genes were cloned from PCR-amplified products of DNA and RNA extracted from a subgingival plaque sample. Approximately 500 bp were amplified in PCRs by using universally conserved primers with polylinker tails. Purified PCR products were cloned into Escherichia coli by using the plasmid vector pUC19. The resultant clone library was screened by colony hybridization with a radiolabeled, treponeme-specific oligonucleotide probe. The 16S rRNA inserts of 81 spirochetal clones were then sequenced by standard procedures. Sequences were compared with 16S rRNA sequences of 35 spirochetes, including the four known cultivable oral treponeme species. The analysis revealed an unexpected diversity of oral treponemes from a single patient. When 98% or greater sequence similarity was used as the definition of a species-level cluster, the clone sequences were found to represent 23 species. When 92% similarity was used as the definition, the clones fell into eight major groups, only two of which contained named species, Treponema vincentii and Treponema denticola, while Treponema pectinovorum and Treponema socranskii were not represented in any cluster. Seven of the 81 spirochetal clones were found to contain chimeric 16S rRNA sequences. In situ fluorescence hybridization with a fluorescein isothiocyanate-labeled oligonucleotide probe specific for one of the new species representing cluster 19 was used to identify cells of the target species directly in clinical samples.

Characterization of a tandemly repetitive DNA sequence from Haemonchus contortus

Genomic DNA from the sheep parasitic nematode Haemonchus contortus was shotgun cloned in the plasmid vector pUC18. Recombinants which gave the strongest hybridization signals to 32P-radiolabelled genomic DNA were selected as representatives of the repetitive component of the parasite DNA. One repetitive sequence which hybridized only with DNA from H. contortus and not with DNA from two other sheep nematodes ( Trichostrongylus colubriformis and Ostertagia circumcincta) was further characterized by sequencing and dot blot analysis. A related repeat was found in the closely related species Haemonchus placei. Experiments to determine the genomic organization of the repeat showed that it existed in a multi-copy number tandem array. This is the first report on the characterization of repetitive DNA in sheep parasitic nematodes.

Molecular cloning of the S-layer protein gene ofCampylobacter rectusATCC 33238

A genomic DNA library of Campylobacter rectus (Wolinella recta) ATCC 33238 was constructed using bacteriophage lambda EMBL3 as a vector. One clone expressing the S-layer protein was identified immunologically with the antiserum to the S-layer protein of C. rectus ATCC 33238. Western immunoblotting using monoclonal antibodies directed against the S-layer protein showed a single blot of recombinant protein at 150 kDa, suggesting that this clone contained the entire coding region of the S-layer protein gene. Additionally, the S-layer protein gene was subcloned into plasmid vector pUC18. Southern hybridization revealed that the S-layer protein gene was present on the chromosome of C. rectus as a single-copy gene, and that there were minor heterogeneities among the S-layer protein genes of clinical isolates. Moreover, a spontaneous stable mutant strain, which has no S-layer expression, also conserved the full-length gene of the S-layer protein.

Molecular cloning of the S-layer protein gene of Campylobacter rectus ATCC 33238

A genomic DNA library of Campylobacter rectus (Wolinella recta) ATCC 33238 was constructed using bacteriophage lambda EMBL3 as a vector. One clone expressing the S-layer protein was identified immunologically with the antiserum to the S-layer protein of C. rectus ATCC 33238. Western immunoblotting using monoclonal antibodies directed against the S-layer protein showed a single blot of recombinant protein at 150 kDa, suggesting that this clone contained the entire coding region of the S-layer protein gene. Additionally, the S-layer protein gene was subcloned into plasmid vector pUC18. Southern hybridization revealed that the S-layer protein gene was present on the chromosome of C. rectus as a single-copy gene, and that there were minor heterogeneities among the S-layer protein genes of clinical isolates. Moreover, a spontaneous stable mutant strain, which has no S-layer expression, also conserved the full-length gene of the S-layer protein.

Cloning and expression of two Pasteurella multocida genes in Escherichia coli

A library of cloned Pasteurella multocida (toxigenic strain 9222, serotype D2) genomic sequences was constructed in Escherichia coli by incorporating TaqI digestion fragments into the plasmid vector pUC19. Immunological screening with antibodies directed against porin H, the major protein of the P multocida outer membrane, allowed the identification of a recombinant plasmid containing a 2.-kbp DNA insert. This plasmid encoded the synthesis of two polypeptides, p25 (25 kDa) and p28 (28 kDa) which were detected in the different compartments of the E coli transformant. The peptide p25 was more abundant in the periplasm whereas p28 was mainly found in the cell envelope and in the cytosol. Immunological analysis indicates that p25, in contrast to p28, is antigenically related to porin H of P multocida. The expression in E coli of the gene encoding p28 was enhanced by induction of the lac promoter.

Detection of mycoplasma bovis using in vitro deoxyribonucleic acid amplification

Detection of Mycoplasma bovis by traditional culture methods is rather time-consuming and is often hampered by bacterial contamination. The development of a rapid and specific alternative is, therefore, an important prerequisite in improving the diagnosis of bovine diseases caused by this agent. The authors have successfully used nucleic acid probes containing genomic restriction fragments of M. bovis cloned into the plasmid vector pUC19 for species-specific detection by dot blot hybridisation of small quantities of M. bovis deoxyribonucleic acid (DNA). The problem of direct M. bovis detection from contaminated milk could not be solved using this procedure. Therefore, further research was conducted using in vitro DNA amplification by polymerase chain reaction (PCR). Species-specific nucleic acid probes were sequenced and suitable PCR primers selected. Using the PCR procedure, ten colony-forming units (CFU) were detected from broth cultures and, after DNA isolation, the equivalent of 1 CFU was detected. Direct detection of M. bovis from biological samples proved extremely difficult due to protein interference. It was shown, however, that direct PCR detection from milk is possible after effective protein removal by combined extraction and protease digestion.

Cloning of the cyclomaltodextrinase gene from Bacillus subtilis high-temperature growth transformant H-17.

The cyclomaltodextrinase gene from Bacillus subtilis high-temperature growth transformant H-17 was cloned on separate PstI, BamHI, and EcoRI fragments into the plasmid vector pUC18, but was expressed in an inactive form in the host, Escherichia coli DH5 alpha. High level constitutive expression of the gene product was also detrimental to the E. coli host, which led to structural instability of the recombinant plasmid. The cyclomaltodextrinase gene was cloned on a 3-kb EcoRI fragment into the plasmid vector pPL708, and the fragment was structurally maintained in the host B. subtilis YB886. The cloned gene product was synthesized in an enzymatically active form in the B. subtilis host; however, expression was at a low level. Subcloning of the 3-kb EcoRI fragment into pUC18 and transformation into E. coli XL1-Blue (F' lacIq) indicated that the cyclomaltodextrinase gene was cloned with its own promoter, since expression of the gene occurred in the absence of IPTG. Subcloning of the cyclomaltodextrinase gene downstream from the Bacillus temperature phage SPO2 promoter of pPL708 may increase expression of this gene.