[3] An integrated and simplified approach to cloning into plasmids and single-stranded phages

Crouse, Gray F.; Frischauf, A.-M.; Lehrach, Hans

doi:10.1016/0076-6879(83)01006-x

Cited by 203 publications

(83 citation statements)

References 8 publications

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“…directly from low-melting-point agarose gels into the indicated vector (8). pDRS6 is the 0.6-kb EcoRI-StuI fragment of the 2.9-kb EcoRI fragment of XCh4ADHFR121 cloned into the EcoRI-HindII site of pEMBL8.…”

Section: Methodsmentioning

confidence: 99%

Analysis of the mouse dhfr promoter region: existence of a divergently transcribed gene.

Crouse¹,

Leys²,

McEwan³

et al. 1985

Mol. Cell. Biol.

103

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The use of murine dihydrofolate reductase (dhfr) gene amplification mutants enabled us to identify important structural and functional features of the dhfr promoter region. We found another transcription unit, at least 14 kilobases in size, which initiates within 130 base pairs of the major dhfr transcript and is transcribed divergently. The 5' ends of both transcripts were analyzed and found to have multiple initiation sites. The major dhfr transcript and the divergent transcript appear to share the same promoter region; the longer transcripts of the dhfr gene overlap with the divergent transcripts and use a different promoter region. The divergent transcript appears to code for a protein; an homologous sequence to its first exon is found in the corresponding location near the human dhfr gene.

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Section: Methodsmentioning

confidence: 99%

Analysis of the mouse dhfr promoter region: existence of a divergently transcribed gene.

Crouse¹,

Leys²,

McEwan³

et al. 1985

Mol. Cell. Biol.

103

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“…Plasmid DNA minipreparations were performed using 1n5 ml of an overnight culture in King's medium B following a modified alkaline lysis procedure (Zhou et al, 1990), and intact plasmids were separated by electrophoresis on 0n6% agarose (Hispanlab) gels in 1i TAE buffer (40 mM Tris\ acetate, 1 mM EDTA, pH 8n0) at 3n2 V cm − " for 5-6 h at room temperature or for about 16 h at 4 mC . Cloning of DNA fragments was done essentially as described by Crouse et al (1983). DNA was introduced into Pseudomonas by electroporation (Keen et al, 1992).…”

Section: Bacterial Strains Plasmids and Growth Conditionsmentioning

confidence: 99%

Phylogeny of the replication regions of pPT23A-like plasmids from Pseudomonas syringae The EMBL accession numbers for the sequences reported in this paper are AJ276998–AJ277021.

2000

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It was previously shown that most Pseudomonas syringae strains contain one or more plasmids with cross-hybridizing replication regions and other areas of homology, and these plasmids were designated the pPT23A-like family. The majority of these plasmids encode genes conferring epiphytic fitness or resistance to antibacterial compounds and those investigated in this study are essential for pathogenicity or increased virulence. The phylogeny of 14 pPT23A-like plasmids from five P. syringae pathovars was studied by comparing a fragment of the sequence of their repA genes (encoding a replicase essential for replication). In the phylogenetic tree obtained, four groups (Z 888% identity between their members) could be identified. The first group contained the plasmids from three P. syringae pv. tomato strains, a P. syringae pv. apii strain and five out of the seven P. syringae pv. syringae strains, with identity ranging between 888 and 100 %. The clustering of the pv. syringae strains did not reflect host specialization or previously reported phylogenetic relationships. The second group contained the plasmids from two strains of pv. glycinea and pv. tomato (955 % identity), and it also included the previously sequenced replicon of a pathogenicity plasmid from P. syringae pv. phaseolicola. The plasmids from the remaining two pv. syringae strains were distantly related to the other plasmid sequences. Hybridization experiments using different genes or transposable elements previously described as plasmid-borne in P. syringae, showed that the gene content of highly related plasmids could be dissimilar, suggesting the occurrence of major plasmid reorganizations. Additionally, the phylogeny of the different native plasmids did not always correlate with the phylogeny of their harbouring strains, as determined by the analysis of extragenic repetitive consensus (ERIC) and arbitrarily primed PCR (AP-PCR) products. Collectively, these results suggest that pPT23A-like plasmids were, in most cases, acquired early during evolution.

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“…Deletion derivatives were constructed by digesting the parent plasmid with one or two endonucleases and ligating the resulting products with T4 DNA ligase. For subcloning, desired DNA fragments were isolated from agarose gels by electroelution (27) or from lowmelting-point agarose gels following electrophoresis (6). Specific DNA fragments were ligated with pBR322, pBR325, or pUC18 DNA previously digested with appropriate restriction endonucleases.…”

mentioning

confidence: 99%

Cloning of pectate lyase gene pel from Pseudomonas fluorescens and detection of sequences homologous to pel in Pseudomonas viridiflava and Pseudomonas putida

Liao

1991

J Bacteriol

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Pectate lyase (PL) depolymerizes pectin and other polygalacturonates (PGAs) and is thought to play a role in bacterial invasion of plants. Production of PL by the soft-rotting pathogen Pseudomonasfluorescens CY091 is regulated by Ca2+. In the presence of Ca2+, this bacterium constitutively synthesizes PL in media containing glucose, glycerol, or PGA and excretes over 87% of total PL into culture fluids. In the absence of Ca2 , the organism fails to use PGA as a carbon source and produces very low levels of PL in media containing glucose or glycerol. Of the small amount of PL produced by the bacterium in Ca2+-deficient media, over 78% was detected within the cells, indicating that Ca2' is critical not only for the production but also for the secretion of PL. The pel gene, encoding an alkaline PL (p1 10.0, Mr 41,000) was cloned and located on the overlapping region of a 4.3-kb Sall and a 7.1-kb EcoRI fragment. Pseudomonas fluorescens is a heterogenous species and consists of a diversity of ecologic and physiological groups (36). Certain strains of this species are postharvest pathogens of plants, which cause soft rot of fruits and vegetables in storage and at markets (25). Soft-rotting P. fluorescens (often referred to as P. marginalis) is capable of degrading pectic components of plant cell walls by producing a wide variety of pectolytic enzymes, including pectin methylesterase (30), pectin lyase (33, 35), polygalacturonase (10,30,42), and pectate lyase (PL) (9,10,12,22,30,42). Production of methylesterase, pectin lyase, and polygalacturonase is rare and has been detected only in a few strains (30,33,35,42). With the exception of one strain (30), all of the soft-rotting pseudomonads so far studied produce PL. Thus, it is generally believed that PL is the principal or sole enzyme responsible for tissue maceration caused by most strains of P. fluorescens and P. viridiflava (22, 24).The PL system of P. fluorescens is considerably different from that of Erwinia spp., although soft-rot symptoms caused by the two organisms are similar. In Erwinia spp., all strains so far studied produce three to five PL isozymes (pl 4.5 to 10.0) (18), whereas in P. fluorescens, all eight strains recently examined in our laboratory produce one or possibly two PLs (22). Furthermore, production of PLs in Erwinia spp. is induced by pectic substrates and subjected to catabolite or self-catabolite repression (18). In P. fluorescens, however, the mode of PL production varies considerably among strains and can be constitutive (30,42) or inducible (9,30,43). In some strains, PL production is induced by pectic substances in a mechanism resembling that previously demonstrated in Erwinia spp. (18). In others, the regulatory factors or mechanisms have not been clearly defined. For example, Zucker and Hankin (43) showed that a nonpectolytic isolate of P. fluorescens can be converted to pectolytic by a series of subcultures in media containing pectin or plant tissue extracts. Hildebrand (10) reported that expression of a pectolytic phenotype in fluo...

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[3] An integrated and simplified approach to cloning into plasmids and single-stranded phages

Cited by 203 publications

References 8 publications

Analysis of the mouse dhfr promoter region: existence of a divergently transcribed gene.

Analysis of the mouse dhfr promoter region: existence of a divergently transcribed gene.

Phylogeny of the replication regions of pPT23A-like plasmids from Pseudomonas syringae The EMBL accession numbers for the sequences reported in this paper are AJ276998–AJ277021.

Cloning of pectate lyase gene pel from Pseudomonas fluorescens and detection of sequences homologous to pel in Pseudomonas viridiflava and Pseudomonas putida

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