Search PubMed⌕ Search

Biomedical subjects

W J Dower

Publications and source records attributed to W J Dower.

32 records · Page 2Linked to original sources
PubMed1994

Membrane insertion defects caused by positive charges in the early mature region of protein pIII of filamentous phage fd can be corrected by prlA suppressors.

The filamentous phage coat protein pIII has been used to display a variety of peptides and proteins to allow easy screening for desirable binding properties. We have examined the biological constraints that restrict the expression of short peptides located in the early mature region of pIII, adjacent to the signal sequence cleavage site. Many functionally defective pIII fusion proteins contained several positively charged amino acids in this region. These residues appear to inhibit proper insertion of pIII into the Escherichia coli inner membrane, blocking the assembly and extrusion of phage particles. Suppressor mutations in the prlA (secY) component of the protein export apparatus dramatically alleviate the phage growth defect caused by the positively charged residues. We conclude that insertion of pIII fusion proteins into the inner membrane can occur by a sec gene-dependent mechanism. The suppressor strains should be useful for increasing the diversity of peptides displayed on pIII in phage libraries.

Amino Acid Sequence↗
PubMed1993

Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP.

We have used affinity panning of libraries of bacteriophages that display random octapeptide or dodecapeptide sequences at the N-terminus of the adsorption protein (pIII) to characterize peptides that bind to the endoplasmic reticulum chaperone BiP and to develop a scoring system that predicts potential BiP-binding sequences in naturally occurring polypeptides. BiP preferentially binds peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions, suggesting that peptides bind in an extended conformation, with the side chains of alternating residues pointing into a cleft on the BiP molecule. Synthetic peptides with sequences corresponding to those displayed by BiP-binding bacteriophages bind to BiP and stimulate its ATPase activity, with a half-maximal concentration in the range 10-60 microM.

Adenosine Triphosphatases↗
PubMed1993

Generation and screening of an oligonucleotide-encoded synthetic peptide library.

We have prepared a library of approximately 10(6) different peptide sequences on small, spherical (10-microns diameter) beads by the combinatorial chemical coupling of both L- and D-amino acid building blocks. To each bead is covalently attached many copies of a single peptide sequence and, additionally, copies of a unique single-stranded oligonucleotide that codes for that peptide sequence. The oligonucleotide tags are synthesized through a parallel combinatorial procedure that effectively records the process by which the encoded peptide sequence is assembled. The collection of beads was screened for binding to a fluorescently labeled anti-peptide antibody using a fluorescence-activated cell sorting instrument. Those beads to which the antibody bound tightly were isolated by fluorescence-activated sorting, and the oligonucleotide identifiers attached to individual sorted beads were amplified by the PCR. Sequences of the amplified DNAs were determined to reveal the identity of peptide sequences that bound to the antibody with high affinity. By combining the capacity for information storage in an oligonucleotide code with the tremendous level of amplification possible through the PCR, we have devised a means for specifying the identity of each member of a vast library of molecules synthesized from both natural and unnatural chemical building blocks. In addition, we have shown that the use of flow cytometry instrumentation permits facile isolation of individual beads that bear high-affinity ligands for biological receptors.

Amino Acid Sequence↗
PubMed1992

Selective enrichment and characterization of high affinity ligands from collections of random peptides on filamentous phage.

Large collections of random peptides can be expressed on the N-terminus of the pIII protein of filamentous phage and screened for binding to antibodies and other receptors. In our previous work with a monoclonal antibody (3E7) (Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-6382, 1990), we showed that a high proportion of the selected peptides had relatively low affinity (Kd's greater than 1 microM). Here we describe conditions for selective enrichment of phage expressing high affinity peptides. This is done by allowing the phage to interact with a low concentration of 3E7 Fab followed by extensive washing to allow dissociation of phage-bearing peptides with low affinity. These affinity selection conditions were applied to the pool of phage previously selected using a high concentration of IgG. A phage clone with the known high affinity ligand YGGFL (Kd 7.1 nM) and several other closely related peptides were isolated. The dissociation rate of 125I-3E7 Fab from several phage clones approximated that of phage expressing YGGFL. A good correlation was found between the dissociation rate of the peptides found on phage and the equilibrium binding constants of chemically synthesized peptides. The strategy of using a low concentration of receptor and extensive washing to select phage-bearing high affinity peptides, combined with assays to determine the specificity and relative affinity of peptides on isolated phage clones, should be generally applicable in using the peptides-on-phage system for discovery of high affinity receptor ligands.

Amino Acid Sequence↗
PubMed1992

Phage power.

Explore the source record for details and available documents.

Journal Article↗
PubMed1990

Peptides on phage: a vast library of peptides for identifying ligands.

We have constructed a vast library of peptides for finding compounds that bind to antibodies and other receptors. Millions of different hexapeptides were expressed at the N terminus of the adsorption protein (pIII) of fd phage. The vector fAFF1, derived from the tetracycline resistance-transducing vector fd-tet, allows cloning of oligonucleotides in a variety of locations in the 5' region of gene III. A library of 3 x 10(8) recombinants was generated by cloning randomly synthesized oligonucleotides. The library was screened for high-avidity binding to a monoclonal antibody (3-E7) that is specific for the N terminus of beta-endorphin (Tyr-Gly-Gly-Phe). Fifty-one clones selected by three rounds of the affinity purification technique called panning were sequenced and found to differ from previously known ligands for this antibody. The striking finding is that all 51 contained tyrosine as the N-terminal residue and that 48 contained glycine as the second residue. The binding affinities of six chemically synthesized hexapeptides from this set range from 0.35 microM (Tyr-Gly-Phe-Trp-Gly-Met) to 8.3 microM (Tyr-Ala-Gly-Phe-Ala-Gln), compared with 7.1 nM for a known high-affinity ligand (Tyr-Gly-Gly-Phe-Leu). These results show that ligands can be identified with no prior information concerning antibody specificity. Peptide libraries are also likely to be useful in finding ligands that bind to other classes of receptors and in discovering pharmacologic agents.

Amino Acid Sequence↗
PubMed1988

High efficiency transformation of E. coli by high voltage electroporation.

E. coli can be transformed to extremely high efficiencies by subjecting a mixture of cells and DNA to brief but intense electrical fields of exponential decay waveform (electroporation). We have obtained 10(9) to 10(10) transformants/micrograms with strains LE392 and DH5 alpha, and plasmids pUC18 and pBR329. The process is highly dependent on two characteristics of the electrical pulse: the electric field strength and the pulse length (RC time constant). The frequency of transformation is a linear function of the DNA concentration over at least six orders of magnitude; and the efficiency of transformation is a function of the cell concentration. Most of the surviving cells are competent with up to 80% transformed at high DNA concentration. The mechanism does not appear to include binding of the DNA to the cells prior to entry. Possible mechanisms are discussed and a simple procedure for the practical use of this technique is presented.

DNA, Bacterial↗
PubMed1988

High-voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA.

Electroporation permits the uptake of DNA by mammalian cells and plant protoplasts because it induces transient permeability of the cell membrane. We investigated the utility of high-voltage electroporation as a method for genetic transformation of intact bacterial cells by using the enteric pathogen Campylobacter jejuni as a model system. This report demonstrates that the application of high-voltage discharges to bacterial cells permits genetic transformation. Our method involves exposure of a Campylobacter cell suspension to a high-voltage exponential decay discharge (5-13 kV/cm) for a brief period of time (resistance-capacitance time constant = 2.4-26 msec) in the presence of plasmid DNA. Electrical transformation of C. jejuni results in frequencies as high as 1.2 x 10(6) transformants per microgram of DNA. We have investigated the effects of pulse amplitude and duration, cell growth conditions, divalent cations, and DNA concentration on the efficiency of transformation. Transformants of C. jejuni obtained by electroporation contained structurally intact plasmid molecules. In addition, evidence is presented that indicates that C. jejuni possesses DNA restriction and modification systems. The use of electroporation as a method for transforming other bacterial species and guidelines for its implementation are also discussed.

Bacteriological Techniques↗
PubMed1984

Gene amplification in a leukemic patient treated with methotrexate.

Resistance to methotrexate (MTX) has been shown in mouse, hamster, and human cell lines grown in sequentially increased MTX concentrations to be due to increased dihydrofolate reductase (DHFR) synthesis and a proportional increase in DHFR gene copy number. Leukemia cells of a patient were studied to assess change in DHFR gene copy number after MTX treatment. The patient presented with chronic myeloid leukemia which rapidly evolved into blast crisis; 90% of peripheral white cells were lymphoblasts. Treatment included intrathecal and intravenous MTX; the lymphoblasts were reduced to undetectable levels. Three months later a second blast crisis occurred; 90% of peripheral white cells were lymphoblasts. Cells from each blast crisis were obtained by leukapheresis and stored for study. Quantification of DHFR gene copy number in DNA from lymphoblasts before and after MTX treatment was done: a radiolabeled cloned cDNA constituting the mouse DHFR coding sequence was used to probe high molecular weight pretreatment and posttreatment DNA bound to nitrocellulose filters. Posttreatment DNA contained two- to three-fold more DHFR gene sequences than pretreatment DNA. Quantitative Southern blotting of EcoRI-digested pretreatment and posttreatment DNA confirmed amplification of the DHFR gene. Karyotype analysis showed no double minute chromosomes or homogeneously staining regions. This is the first study demonstrating DHFR gene amplification in leukemia cells sampled in vivo from a patient treated with MTX.

Adult↗
PubMed1971

Aplysia californica: analysis of nuclear DNA in individual nuclei of giant neurons.

The nuclei of the giant neurons of the marine mollusk Aplysia californica can contain more than 0.2 microgram of DNA. This is more than 200,000 times as much DNA as the haploid amount found in Aplysia sperm. On the basis of nuclear DNA content, the giant neurons R-2, P-1, and L-6 of adult animals can each be divided into at least two populations. The mean DNA content of these two populations (0.067 and 0.131 microgram of DNA) are approximately related by a factor of 2. This suggests that much and perhaps all of the genome replicates repeatedly (up to 16 times) during the growth and development of these neurons and that each replication is synchronous. The enormous amount of DNA in these cells opens up the possibility of characterizing the DNA and other constituents of chromatin from individual but phenotypically different neurons.

Animals↗