Rapid and efficient cosmid cloning

Ish‐Horowicz, David; Burke, Julian F.

doi:10.1093/nar/9.13.2989

Cited by 1,751 publications

(659 citation statements)

References 21 publications

(17 reference statements)

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“…DNA fragments to be sequenced were subcloned into M13mp18 or mp19 (60), using standard procedures (4), with Escherichia coli TG1 (16) used as the host strain. Doublestrande M13 DNA was prepared (25), and partial deletions of the cloned fragments were generated by using exonuclease III (Erase-a-Base system; Promega) or by using restriction enzymes which cleaved the DNA within the insert and within the M13 polylinker. Oligonucleotide primers were used to sequence regions not covered by these strategies; these were synthesized by using a model 180B DNA synthesizer (Applied Biosystems, Inc., Foster City, Calif.) or were purchased from Macromolecular Resources (Colorado State University).…”

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confidence: 99%

Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases

1995

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Pseudomonas aeruginosa secretes a fluorescent siderophore, pyoverdine, when grown under iron-deficient conditions. Pyoverdine consists of a chromophoric group bound to a partly cyclic octapeptide. As a step toward understanding the molecular events involved in pyoverdine synthesis, we have sequenced a gene, pvdD, required for this process. The gene encodes a 2,448-residue protein, PvdD, which has a predicted molecular mass of 273,061 Da and contains two highly similar domains of about 1,000 amino acids each. The protein is similar to peptide synthetases from a range of bacterial and fungal species, indicating that synthesis of the peptide moiety of pyoverdine proceeds by a nonribosomal mechanism. The pvdD gene is adjacent to a gene, fpvA, which encodes an outer membrane receptor protein required for uptake of ferripyoverdine.Pseudomonas aeruginosa is an opportunistic pathogen which infects injured, immunodeficient, or otherwise compromised patients. The bacteria secrete a siderophore, pyoverdine, which is likely to play an important role in infection by competing with transferrin for iron in order to overcome the iron-withholding defense mechanism present in mammals (3,11,42). Almost all isolates of P. aeruginosa from infected patients secrete pyoverdine (9,22,29), and it has been shown that pyoverdine is present in the sputa of cystic fibrosis patients infected with P. aeruginosa (21). A mutant of P. aeruginosa which is unable to synthesize pyoverdine showed reduced virulence in an animal model of infection (24).Pyoverdine from P. aeruginosa PAO is a water-soluble, yellow-green fluorescent compound. It consists of a dihydroxyquinoline chromophoric group linked to an eight-residuevia the N-terminal serine, with a small dicarboxylic acid attached to the chromophore. Iron complexation is thought to occur through oxygen atoms present on the dihydroxyquinoline and two hydroxamate units supplied by the L-N 5 -OH-Orn residues. Several Pseudomonas species produce similar compounds, variously called pyoverdines or pseudobactins, all of which have the same dihydroxyquinoline group but differ in the nature of the attached peptide (reviewed in references 1 and 7), and it is likely that all of these are synthesized by similar mechanisms. Synthesis of the chromophoric group begins with condensation of D-tyrosine and L-2,4-diaminobutyric acid (7), with glutamic acid being the precursor of the amide-linked dicarboxylic acid (50). It has been suggested that biosynthesis of the peptide moiety of pyoverdine occurs by a nonribosomal mechanism (30).Little is known about the molecular nature of enzymes involved in the biosynthesis of pyoverdine in P. aeruginosa or any other pseudomonad. Recently, pvdA, which encodes an enzyme (L-ornithine N 5 -oxygenase) responsible for catalyzing the hydroxylation of ornithine, an early step in pyoverdine biosynthesis in P. aeruginosa, has been characterized at the molecular level (55). The pbsC gene, which is involved in synthesis of pseudobactin by Pseudomonas sp. strain M114, has also been seque...

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Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases

1995

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“…The second method involved growing plasmid-harboring RR1 or JM107 cells to saturation followed by a scaled up (100 ml) Ish-Horowicz and Burke (22) method of purification and equilibrium sedimentation in CsCl gradients. After dialysis against 0.2 mM EDTA (pH 7.0), the yield of plasmid was determined by A260 readings.…”

Section: Methodsmentioning

confidence: 99%

Xrep, a plasmid-stimulating X chromosomal sequence bearing similarities to the BK virus replication origin and viral enhancers

Riley¹,

Reeves

Gartler³

1986

Nucl Acids Res

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“…For Southern blot analysis, 2 lag genomic DNA was used per restriction digest. Plasmid DNA was purified according to a modified small-scale method [23].…”

Section: Strains Plasmids and Dna Preparationsmentioning

confidence: 99%

Identification and characterization of a lower plant Ypt/Rab guanosine dissociation inhibitor (GDI)

Beyser

Fabry

1996

FEBS Letters

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Rapid and efficient cosmid cloning

Cited by 1,751 publications

References 21 publications

Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases

Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases

Xrep, a plasmid-stimulating X chromosomal sequence bearing similarities to the BK virus replication origin and viral enhancers

Identification and characterization of a lower plant Ypt/Rab guanosine dissociation inhibitor (GDI)

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