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Biomedical subjects

W Mandecki

Publications and source records attributed to W Mandecki.

At least 37 records · Page 2Linked to original sources
PubMed1991

C5a structural requirements for neutrophil receptor interaction.

A number of C5a modifications were tested to determine effects on receptor binding to polymorphonuclear leukocyte (PMNL) membrane receptors and triggering of PMNL chemokinesis and myeloperoxidase (MPO) release. Site-directed mutagenesis was used to probe relationships of key C-terminal residues, and suggested a role for additional sites, particularly Lys19-20. A synthetic peptide based on C5a 19-30, weakly inhibited C5a binding. Potency of the C-terminal octapeptide, a full agonist, was markedly improved by a single Phe substitution for His67, and a Phe point mutation at this site was shown to enhance activity of the full recombinant protein.

Cells, Cultured↗
PubMed1990

A totally synthetic plasmid for general cloning, gene expression and mutagenesis in Escherichia coli.

A first totally synthetic Escherichia coli plasmid has been designed, constructed and shown to be a functional, stable, high-copy cloning vector. The FokI method of gene synthesis [Mandecki and Bolling, Gene 68 (1988) 101-107] was used to assemble the plasmid from 30 oligodeoxyribonucleotides. The plasmid contains synthetic modules for the beta-lactamase-encoding gene (bla), replication origin, lacZ gene fragment and multicloning site. The plasmid is patterned after the pUC-type plasmids and has a copy number similar to that of pUC plasmids. The major changes introduced include the removal of nearly 50% of the restriction sites present in pUC plasmids, reduction of plasmid size to 2050 bp, and introduction of transcription terminators downstream from both the bla gene and lacZ fragment. The changes facilitate a number of techniques, such as cloning, mutagenesis, expression and restriction analysis.

Base Sequence↗
PubMed1990

A method for construction of long randomized open reading frames and polypeptides.

A method is presented for construction of randomized open reading frame sequences (ORFs) and gene libraries containing them. The building blocks for the ORFs were 75 bp long DNA fragments generated by cloning sequences from a single synthetic oligonucleotide preparation by bridge mutagenesis. The fragments had the property that, regardless of their orientation in the ligated product, the ORF of the construct was maintained. The heterogeneity of the ORFs resulted from the random ligation of 2000 different DNA fragments. The randomized ORFs were cloned downstream from the lac promoter in a multicopy plasmid in Escherichia coli. To test the method, a library of 10(6) clones was constructed.

Amino Acid Sequence↗
PubMed1990

An Escherichia coli expression vector for high-level production of heterologous proteins in fusion with CMP-KDO synthetase.

The construction of a vector which overproduces the enzyme, CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl-transferase (CMP-KDO synthetase or CKS) and its use as an expression vector for producing heterologous proteins in E. coli is described. The vector, which includes a modified lac promoter and synthetic ribosome binding site upstream of the native kdsB gene (encoding CKS), produces CKS at levels as high as 70% of the total cellular proteins. Several heterologous gene sequences have been fused to the 3'-end of the kdsB gene with resulting protein fusions produced at a level of up to 40% of the total cellular proteins.

Base Sequence↗
PubMed1990

Simple biological assay for the error rate upon cloning of synthetic oligodeoxyribonucleotides.

A method was developed for determination of the rate of undesired point mutations upon cloning of synthetic DNA. The method relies on cloning of an oligonucleotide(s) into the E. coli alkaline phosphatase gene inactivated due to a small deletion within the active site. The oligonucleotide adds back the deleted sequence, but simultaneously introduces a missense mutation at a critical position. The activity of the enzyme is restored only if there is a predefined sequence change within the codon specifying an essential residue of the active site. The clones carrying the reactivated gene are detected by colony color screening on plates. The method is fast and simple, does not require specialized equipment nor enzymatic reactions, although a separate oligonucleotide needs to be provided for each sequence change to be evaluated. The procedure allows for the use of crude extracts of oligonucleotides and distinguishes between different types of sequence changes.

Alkaline Phosphatase↗
PubMed1989

Identification of receptor-binding residues in the inflammatory complement protein C5a by site-directed mutagenesis.

C5a is an inflammatory mediator potentially involved in a number of diseases. To help define which of its 74 residues are important for receptor binding and response triggering, changes in the amino acid sequence of C5a were introduced by site-directed mutagenesis. Synthetic C5a-encoding genes incorporating point mutations were expressed in Escherichia coli, and the mutant proteins were purified to homogeneity. Modifications of the C5a molecule causing parallel reductions in binding to polymorphonuclear leukocyte membranes and in stimulation of polymorphonuclear leukocyte locomotion (chemokinesis) suggest that carboxyl-terminal residues Lys-68, Leu-72, and Arg-74 interact with the receptor. Substitutions in the disulfide-linked core of C5a revealed involvement of Arg-40 or nearby residues, because potency losses were associated with only localized conformational changes as detected by NMR. Surprisingly, a substitution at core residue Ala-26, which did not alter C5a core structure, appeared from NMR results to reduce potency by causing a long-distance conformational change centered on residue His-15. Thus, at least three discontinuous regions of the C5a molecule appear to act in concert to achieve full potency.

Amino Acid Sequence↗
PubMed1988

FokI method of gene synthesis.

An accurate, fast and simple method is presented for synthesis of a gene, or any DNA fragment with a defined sequence. The method is based on the observation that large (approx. 100 bp long) inserts can be cloned into a plasmid using a technique of oligodeoxynucleotide (oligo)-directed double-strand (ds) break repair. The procedure involves transformation of Escherichia coli with a denatured mixture of an insert-carrying oligo and linearized plasmid DNA [Mandecki, Proc. Natl. Acad. Sci. USA 83 (1986) 7177-7181]. The nucleotide (nt) sequences are inserted between two FokI restriction nuclease sites in one of four pUC-derived plasmids. Since FokI makes a staggered ds break at a DNA site 9 and 13 nt away from its recognition site, upon cleavage of the plasmid DNA with FokI, a restriction fragment is liberated that by design contains unique 4-nt-long 5'-protruding ends. The uniqueness of ends permits efficient and directed simultaneous ligation of several restriction fragments to form a gene. The method offers flexibility due to the modular-type assembly and does not require any restriction sites within the constructed gene. The sequence error rate is low: about one error per 4000 bp of DNA cloned. Synthetic DNA for only one DNA strand needs to be provided. The method was applied to the synthesis of a gene fragment encoding the N-terminal 143 amino acid residues of the human immunodeficiency virus transmembrane protein (p41).

Amino Acid Sequence↗
PubMed1988

Gene synthesis by serial cloning of oligonucleotides.

A rapid and simple method of gene synthesis is presented. A gene is constructed by serial additions of individual gene fragments in the 5'----3' direction. The vector used as the synthesis vehicle contains a unique Bsm I site at the amino terminus of the lacZ gene. Plasmid linearized with Bsm I is recircularized in vivo by oligonucleotide-directed double-strand break repair. The synthetic oligonucleotide used to "bridge" the double-strand break carries a 70- to 100-nucleotide insert which constitutes a portion of the gene along with a BsmI site at the 3' end that regenerates the site and allows for another consecutive round of mutagenesis to extend the gene sequence. The process is repeated until the entire gene is assembled. The method uses the beta-galactosidase color assay as a means of screening for correct insert lengths. The only in vitro enzymatic step necessary is a single Bsm I restriction digest of plasmid DNA. No ligation reactions are required. Only one strand of a gene sequence needs to be synthesized chemically. The gene synthesis method presented here was used to construct the human anaphylatoxin complement factor C3a gene.

Amino Acid Sequence↗
PubMed1987

Crystals of the trp repressor-operator complex suitable for X-ray diffraction analysis.

Crystals of a simulated trp repressor-operator complex have been grown that are large enough and are sufficiently well ordered and durable to provide a high quality molecular image of this regulatory protein X DNA complex to better than 3-A resolution. The "operator" consists of a 2-fold rotationally symmetric 18-base pair duplex that is extended by a dT residue at both 5'-termini. This system exhibits extensive crystal polymorphism. The crystal form and diffraction properties are very sensitive to the length and terminal structure of the operator fragment, as well as the type and concentration of multivalent ions. When combined with the experience reported by others, our results do not support a consistent strategy for crystallization of protein X DNA complexes.

Bacterial Proteins↗
PubMed1987

High-level C5a gene expression and recovery of recombinant human C5a from Escherichia coli.

Poor expression of a synthetic gene for the inflammatory mediator, C5a, was observed in E. coli grown in rich media. Varying the media composition markedly improved expression, although C5a levels still declined rapidly at the end of log phase. Using a protease-deficient strain, C5a was recovered at stationary phase in high yield (13 mg/liter of culture). Recovery was dependent on guanidinium hydrochloride extraction to solubilize the protein and glutathione treatment to promote correct folding. Two-thirds of the C5a retained an amino-terminal methionine. Both forms of recombinant C5a had activity similar to serum-derived C5a in binding to human neutrophil receptors and inducing chemotaxis. The 700-fold improvement in yield made it feasible to obtain gram amounts of C5a and provides an efficient system for site-directed mutagenesis.

Cloning, Molecular↗
PubMed1986

High-level expression of a gene encoding the human complement factor C5a in Escherichia coli.

The synthetic C5a gene was initially found to be expressed poorly in Escherichia coli. We undertook studies to determine the reasons for poor expression and to increase expression. The work was focused on the role of the mRNA structure in C5a expression and stability of its product in E. coli. We present data on the effects of varying the sequence at the 5' end of mRNA as well as different ribosome-binding sites on expression. Evaluation of the stability of C5a showed rapid degradation of C5a in wild-type E. coli (half-life 3-5 min). Screening of several protease-deficient strains of E. coli showed that C5a was much more stable in an htpR strain carrying a mutation in the sigma subunit of RNA polymerase that is specific for heat shock promoters. The mutation is associated with a proteolytic deficiency. The half-life of C5a was increased to 20 min. By manipulating the expression vector, the regulatory region for the C5a gene, the host strain, growth conditions and methods for recovering the protein, C5a levels were increased 300-fold over previously reported amounts to about 3% of total cellular protein.

Base Sequence↗
PubMed1986

Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis.

A DNA double-strand break can be efficiently repaired in Escherichia coli if an oligodeoxyribonucleotide is provided to direct the repair. The oligonucleotide must be at least 20 residues long and have a sequence identical to sequences flanking the break. The phenomenon can be used to introduce defined mutations into DNA in the area of a double-strand break. To obtain mutants, the oligonucleotide that carries a mutation and the denatured linearized plasmid DNA are introduced into E. coli by transformation. No enzymatic manipulation in vitro is required. The mutants can constitute up to 98% of the total number of transformants obtained. The efficiency of mutagenesis decreases as the distance between the mutation and the plasmid cleavage site increases. The universality of the method was tested by introducing mutations into four genes, using four plasmids and three E. coli strains, as well as eight restriction enzymes to linearize DNA. Several models of the oligonucleotide-directed DNA double-strand break repair are discussed.

Base Sequence↗
PubMed1986

A quantitative model for nonrandom generalized transduction, applied to the phage P22-Salmonella typhimurium system.

A mathematical model for nonrandom generalized transduction is proposed and analyzed. The model takes into account the finite number of transducing particle classes for any given marker. The equations for estimation of the distance between markers from contransduction frequency data are derived and standard errors of the estimates are given. The obtained relationships depend significantly on the number of classes of transducing fragments. The model was applied to estimate the number of transducing fragment classes for a given marker in transduction with phage P22 of Salmonella typhimurium. It was found that the literature data on frequencies of contransduction in crosses with mutual substitution of selective and nonselective markers can be rationalized most accurately by assuming that the mean number of classes is equal to 2. An improved method for analysis of cotransduction data is proposed on the basis of our model and the results of calculation. The method relies on solving a set of algebraic equations for cotransduction frequencies of markers located within one phage length. The method allows a relatively precise determination of distances between markers, positions of transducing particle ends and deletion or insertion lengths. The approach is applied to the trp-cysB-pyrF and aroC-hisT-purF-dhuA regions of the Salmonella typhimurium chromosome.

Chromosome Deletion↗
PubMed1985

Chemical synthesis of a gene encoding the human complement fragment C5a and its expression in Escherichia coli.

A gene coding for the C5a fragment of the fifth component of human complement has been chemically synthesized, cloned, and expressed in Escherichia coli. The 253-base-pair gene fragment was built through a two-step enzymic assembly of 16 oligonucleotides, the average length of each being 32 residues. The oligonucleotides were synthesized by using the phosphoramidite method. The gene was cloned in a pBR322-derivative plasmid downstream from the lac up-promoter mutant, UV5-D. The expression of C5a was detected and measured by immunoassay and a radioligand binding assay. C5a from E. coli was comparable to C5a purified from human serum in inhibiting binding of human 125I-labeled C5a to its putative receptor on polymorphonuclear leukocytes. Studies of smooth muscle contraction in isolated guinea pig ileum showed that the recombinant C5a was biologically active and produced cross-tachyphylaxis with human serum-derived C5a. The results demonstrate the feasibility of expressing C5a anaphylatoxin in bacteria and provide a system for mutagenesis of the C5a protein.

Animals↗
PubMed1985

lac Up-promoter mutants with increased homology to the consensus promoter sequence.

Four lac promoter mutants were constructed. The mutations increased the homology between the lac promoter and the consensus promoter sequences by introducing the consensus -10 and -35 regions and the consensus spacing of 17 residues between these two regions. The promoter mutants were cloned into a pBR322-derivatized vector upstream from the lacZ gene, and levels of beta-galactosidase were an indication of promoter activity. All mutants exhibited higher activity than did the wild-type promoter.

Base Sequence↗
PubMed1984

Oligonucleotide mutagenesis of the lacPUV5 promoter.

Synthetic oligonucleotides were used to introduce mutations into the lacPUV5 promoter. Four mutations were obtained at positions -13, -14, and -15, with respect to the transcriptional start site. The effects of these mutations were measured in vivo and the results are discussed with respect to the consensus sequence and other promoter mutations located in this region.

Base Composition↗
PubMed1984

Mutants of the lac promoter with large insertions and deletions between the CAP binding site and the -35 region.

A set of the lac promoter mutants that have varying lengths of the spacer between the CAP binding site and the -35 region was constructed. The mutants have the spacer length increased by five (I5 mutant), or eleven (I11) residues or decreased by eleven residues (D11). We also present a construction of the hybrid between the gal and lac promoters in which the CAP binding site and the -35 region of the gal promoter are fused to the lac -10 region. The promoter fragments were assembled through ligations of chemically synthesized oligodeoxynucleotides and cloned into a pBR322-derivative vector. The results of the in vivo assays of promoter activity show that the I11 mutation results in an active but weak promoter that can be stimulated by CAP, though to a lesser degree than the wild-type lac promoter. The other mutants exhibit no promoter activity. Since the insertion of 11-bp preserves the location of the CAP binding site on the same side on the DNA helix, the data demonstrate the importance of spatial alignment between the CAP binding site and the promoter. The fact that the gal::lac hybrid is inactive as a promoter indicates also that catabolite activation is a highly complex process in which the -35 and -10 regions cannot be easily exchanged between promoters.

Base Sequence↗