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P W Tucker

Publications and source records attributed to P W Tucker.

At least 19 recordsLinked to original sources
PubMed1997

NonO enhances the association of many DNA-binding proteins to their targets.

NonO is an unusual nucleic acid binding protein not only in that it binds both DNA and RNA but that it does so via functionally separable domains. Here we document that NonO enhances the binding of some (E47, OTF-1 and OTF-2) but not all (PEA3) conventional sequence-specific transcription factors to their recognition sites in artificial substrates as well as in an immunoglobulin VHpromoter. We also show that NonO induces the binding of the Ku complex to DNA ends. Ku has no known DNA sequence specificity. These enhancement of binding effects are NonO concentration dependent. Using the E box activity of E47 as a model, kinetic studies demonstrate that the association rate of the protein-DNA complex increases in the presence of NonO while the dissociation rate remains the same, thereby increasing the sum total of the interaction. Oligo competition experiments indicate that NonO does not contact the target DNA in order to enhance the binding activity of DNA binding proteins. Rather, methylation interference analysis reveals that the induced E47 binding-activity has the same DNA-binding sequence specificity as the normal binding. This result suggests that one of the effects of NonO is to induce a true protein-DNA interaction. In this way, it might be possible for NonO to play a crucial role in gene regulation.

Antigens, Nuclear↗
PubMed1996

Analysis and comparison of the mouse and human immunoglobulin heavy chain JH-Cmu-Cdelta locus.

We report here 23,686 bases of contiguous DNA sequences from the mouse germline immunoglobulin heavy chain (H) constant (C) mu delta region. The sequence spans the joining (JH) regions, the mu constant region (C mu), the delta constant region (C delta) coding regions, a domain relic, the mu switch region (S mu), seven blocks of simple sequence repeats, a large unique sequence inverted repeat, a large unique sequence forward repeat, and all of the intervening material. A comparison of this 23.7-kb region with the corresponding human C mu/C delta region reveals clear homology in the coding and introns of C mu but not in the 5' flanking J gene segments nor in the intergenic and C delta regions. This mixed pattern of similarity between the human and the mouse sequences contrasts with high levels of similarity found in the T-cell receptor C alpha/C delta region and alpha and beta myosin genes and the very low levels found in the gamma-crystallin, XRCC1, and beta-globin gene clusters. The human and mouse comparison further suggests the incorporation of novel sequences into expressed genes of IgD.

Animals↗
PubMed1996

Ku is a general inhibitor of DNA-protein complex formation and transcription.

Ku is a ubiquitous and abundant DNA binding protein. Recently, it has been shown that Ku plays a crucial role in double stranded-DNA (dsDNA) break repair such as occurs during the V(D)J recombination of Ig genes. Ku has also been found to provide DNA binding activity to the catalytic domain of DNA-PK which is known to phosphorylate several transcription factors, suggesting that Ku is a multifunctional protein that participates as a component of several functional DNA-protein complexes. Here, we examined the interaction of Ku with several DNA binding proteins. Firstly, the DNA binding interaction between Ku and well-characterized transcription factors (OTF-1, Sp-1, AP-1) was analysed by EMSA. Although sequence non-specific, Ku was strongly competitive with these sequence specific transcription factors on compatible DNA elements, displacing them because of its high affinity association with DNA ends. Secondly, to determine whether this competitive effect was functionally relevant, we tested Ku in an in vitro transcription system with the adenovirus major late promoter. We found that Ku inhibited transcription from linear, but not from circular template DNA. These results suggest that Ku inhibits transcription when it is able to bind to template DNA and that the inhibition is the result of Ku displacing specific transcription factors from DNA.

Antigens, Nuclear↗
PubMed1996

A comparison of 2 analgesic regimens for the control of postoperative periodontal discomfort.

A single blind study of 24 patients compared the postoperative periodontal pain relief and adverse effects associated with a pretreatment regimen with etodolac, a nonsteroidal anti-inflammatory drug (NSAID), to a typical pro re nada (prn) regimen with a combination of acetaminophen with hydrocodone. Patients selected required one or more periodontal osseous surgeries that were judged to involve relatively similar degrees of surgical manipulation. Patients in the etodolac group received two 300 mg capsules 30 minutes prior to surgery and then redosed themselves prn. Patients who received the combination drug were not premedicated and followed a prn regimen. The subjects used a verbal analogue scale to report levels of pain hourly for the first 8 hours (starting 30 minutes prior to surgery) and also indicated any side effects experienced during the first week after surgery. Specific parameters monitored were the mean sum of hourly pain scores, mean hourly pain scores, time to first medication, number of postoperative doses, and adverse effects. Of the parameters studied, the only one that showed a statistically significant difference was the time to first medication. The time span from 30 minutes prior to the beginning of surgery to the first postsurgical dose was greater for etodolac than for the combination drug. However, the total number of medications taken under both regimens was similar. The side effects were minimal for both of the drugs studied. It was concluded that the analgesic regimens tested under clinical practice conditions were comparable in providing analgesia with minimum side effects in uncomplicated periodontal osseous surgery. Studies with larger numbers of patients are needed to definitively address whether these regimens are truly equivalent.

Acetaminophen↗
PubMed1995

The immunoglobulin heavy-chain matrix-associating regions are bound by Bright: a B cell-specific trans-activator that describes a new DNA-binding protein family.

B lymphocyte-restricted transcription of immunoglobulin heavy-chain (IgH) genes is specified by elements within the variable region (VH) promoter and the intronic enhancer (E mu). The gene encoding a protein that binds a VH promoter proximal site necessary for induced mu-heavy-chain transcription has been cloned. This B-cell specific protein, termed Bright (B cell regulator of IgH transcription), is found in both soluble and matrix insoluble nuclear fractions. Bright binds the minor groove of a restricted ATC sequence that is sufficient for nuclear matrix association. This sequence motif is present in previously described matrix-associating regions (MARs) proximal to the promoter and flanking E mu. Bright can activate E mu-driven transcription by binding these sites, but only when they occur in their natural context and in cell lines permissive for E mu activity. To bind DNA, Bright requires a novel tetramerization domain and a previously undescribed domain that shares identity with several proteins, including SWI1, a component of the SWI/SNF complex.

Amino Acid Sequence↗
PubMed1995

Analysis of direct and inverted DJH rearrangements in a human Ig heavy chain transgenic minilocus.

D to JH rearrangement can occur either by a deletional or an inversional mechanism. In this study, we have analyzed deletional and inversional D to JH recombination in a human Ig heavy chain transgenic minilocus. The analysis of these events in a transgenic minilocus rather than in vivo in the human is simplified by the presence of a limited number of well defined VH, D, and JH gene segments in the minilocus. We show that in the transgenic minilocus, all D gene segments can be rearranged by deletion and virtually all can be rearranged by inversion. We also show that depending upon the D gene segment, rearrangement by deletion occurs approximately 1 to 1000 times more frequently than rearrangement by inversion. Our data suggest that in vivo, signal and coding end sequences are the major influences on the rearrangement frequency of a particular gene segment in a given orientation rather than its position within the transgenic locus. Additionally, our data indicate that intronic and recombination signal sequences are involved in the bias for deletion over inversion rather than the recombinase machinery itself.

Animals↗
PubMed1995

Ig gamma 2b transgenes promote B cell development but alternate developmental pathways appear to function in different transgenic lines.

Analysis of B cell development in three strains of gamma 2b transgenic mice shows that the gamma 2b H chain can replace the microH chain in promoting B cell differentiation. The 348C line produces 90% gamma 2b-only B cells and 10% B cells; which co-express gamma 2b and endogenous sIgM and sIgD. These IgG2b+ B cells develop into mature, recirculating CD23+ B cells. The 343-1 and gamma 2b-T15 transgenic mice produce sIgMhigh:sIgDlow:CD23- B cells that generally co-express the gamma 2b transgene-encoded H chain. Such B cells are either developmentally arrested immature B cells or arise from B-1 (CD5) progenitors. The gamma 2b-T15 mice can produce gamma 2b-only CD23+ B cells following inactivation of the endogenous mu locus, whereas 343-1 mice fail to develop B cells. Thus, gamma 2b H chains: 1) can act alone to promote the development of mature B cells, 2) synergize with microH chains for allelic exclusion, and 3) vary in their influence on B cell development in different transgenic mouse strains.

Animals↗
PubMed1995

Directional cloning of an oligonucleotide fragment into a single restriction site.

Oligonucleotide fragments can be directionally subcloned into vectors at a single restriction site. By using T4 DNA polymerase exonuclease activity to treat vector DNA, single-stranded ends can be generated. The oligonucleotide sequences are designed to have sequence complementary to these single-stranded ends. Through the homologous annealing of oligonucleotides to the treated vector ends, the successfully subcloned molecules forms a circular recombinant DNA that is ready for transformation. There is no sequence restriction at the ends of the DNA fragment. All restriction site ends are accessible to this method. This approach for oligonucleotide fragment insertion and together with our previously described general method of exonuclease induced DNA subcloning provide convenient methods for the construction of recombinant DNA.

Amino Acid Sequence↗
PubMed1995

IgD class switching: identification of a novel recombination site in neoplastic and normal B cells.

IgD on normal B lymphocytes usually is co-expressed with IgM. A minority of normal plasma cells and rare B cell malignancies express exclusively IgD (IgM-IgD+). The low frequency has been explained by the lack of a recognizable switch region within the C mu-C delta intron. We analyzed four cases of IgM-IgD+ hairy cell leukemia (HCL) by Southern (DNA) blot analysis and identified two cases with a recombinatorial event within the C mu-C delta intron and deletion of C mu. DNA sequence analysis of junctional regions showed that S mu or the immediate upstream region was used as a donor site and that the C mu-C delta intronic sigma delta region was used as acceptor site. Using polymerase chain reaction, we subsequently analyzed whether similar S mu-sigma delta recombinations occur in normal tonsils containing IgM-IgD+ plasma cells. Multiple products with a size range of 200-800 base pairs were detected in all four individuals, suggesting clustering of acceptor sites within sigma delta. Sequence analysis of three cloned products showed S mu-sigma delta recombinations similar those observed in HCL. The sigma delta region contains a relatively high content of pentameric repeats with an extremely G-rich area and appears to function as a vestigial switch recombination site in normal and neoplastic IgM-IgD+ B cells.

B-Lymphocytes↗
PubMed1994

Mutations within the NH2-terminal transmembrane domain of membrane immunoglobulin (Ig) M alters Ig alpha and Ig beta association and signal transduction.

Potentiation of initial signal transduction events through the cross-linking of the B cell antigen receptor complex appears to be dependent upon the association of membrane immunoglobulin (mIg) with Ig alpha and Ig beta. We made two groups of mutations within the COOH terminus of mIgM substituting: 1) the spacer, transmembrane, and cytoplasmic domains and 2) the NH2-terminal 2-8 amino acids within the transmembrane domain (NLWTTAST). We then evaluated the ability of the mutated receptors to associate with Ig alpha and Ig beta and to initiate signal transduction events (Ca2+ mobilization and phosphorylation by tyrosine protein kinases) after cross-linking mIgM receptors. Mutant mIgM receptors containing substitutions of gamma 2b (spacer, transmembrane, and cytoplasmic domains), AA for TT, and AAAAA for TTAST bound Ig alpha and Ig beta and initiated signal transduction events after mIgM receptor cross-linking. However, substitutions of I-A alpha (spacer, transmembrane, and cytoplasmic domains) or TTVVCALGL for NLWTTAST blocked association of Ig alpha and Ig beta and initiation of signal transduction events. Results indicate that residues within the first 8 amino acids of the transmembrane domain other than TTAST are necessary for receptor function and association with Ig alpha and Ig beta.

Amino Acid Sequence↗
PubMed1994

Production and characterization of recombinant human Ku antigen.

Ku is an ubiquitous nuclear heterodimeric protein consisting of p70 and p86 subunits that binds double-stranded DNA termini and associates with chromosomes in vivo. It was originally described as an autoantigen in patients with certain autoimmune diseases. The individual subunits of Ku have been difficult to isolate from human cells without denaturation and attempts to produce functional recombinant Ku have been largely unsuccessful. Here, we utilize two recombinant baculoviral vectors that carry p70 or p86 cDNA and express the Ku subunits individually as well as assemble them into the complete Ku heterodimer. In an electrophoretic mobility shift assay, recombinant Ku binds to linear double-stranded DNA but not to supercoiled, nicked circular, nor linear single-stranded DNA. Neither subunit binds DNA by itself indicating that heterodimerization is essential for function. We also describe a simple purification method for the isolation of highly purified recombinant Ku using a hexahistidine tag. The baculovirus expression system provides a stable and efficient source of not only the p70 and p86 subunits but also the functional Ku heterodimer.

Animals↗
PubMed1994

Biased utilization of DHQ52 and JH4 gene segments in a human Ig transgenic minilocus is independent of antigenic selection.

We have assessed the effect of antigenic selection on human Ig heavy chain D and JH gene segment utilization in mice that contain transgenes composed of 2 VH (psi VH3-105 and VH5-251), 10 D, 6 JH, C mu, and C gamma 1 human gene segments. Human heavy chains using the functional VH5-251 gene segment are expressed in the serum and on the surface of murine B cells. The second VH gene segment (psi VH3-105) is not expressed as a protein but is rearranged and transcribed into mRNA. We previously reported that the functional VH5-251 mu transcripts preferentially used the DHQ52 and JH4 gene segments similar to their use in the human repertoire. Here, we demonstrate that the nonfunctional (psi VH3-105) gene segment shows the same bias in D and JH gene segment utilization. Because transcripts using the pseudo-VH gene segment cannot be subjected to antigenic selection, we conclude that the restricted repertoire observed is Ag independent. Analysis of pseudo VH gene segment recombination products reveals no bias in D gene segment reading frame utilization. Finally, we demonstrate that in the human transgene coding sequence complementarities can affect the recombination site and that D inversion is common.

Animals↗
PubMed1994

Surface immunoglobulins mediate efficient transport of antigen to lysosomal compartments resulting in enhanced specific antigen presentation by B cells.

A BCL1 immunoglobulin (Ig) transfectant, expressing wild-type surface (s)IgM with the TEPC-15 idiotype (T15-Id) and anti-phosphorylcholine (PC) specificity, was previously shown to present PC-conjugated hen egg-white lysozyme (PC-HEL) to a HEL-specific T cell hybridoma at a lower antigen (Ag) concentration than that required for native HEL. Two variant Ig transfectants, expressing T15-Id sIgM with substitutions either in the entire spacer, transmembrane (TM) domain and cytoplasmic tail (B186 variant) or in the NH2-terminal third of TM domain only (TM2 variant), failed to display this sIgM-mediated, enhanced presentation of PC-HEL at low concentrations. However, prolonged treatment with anti-T15-Id monoclonal antibody (mAb) led to a reduction of surface expression of the T15-Id sIgM in the wild-type and TM2 variant, but not in the B186 variant sIgM transfectants. Treatment with anti-T15-Id mAb also resulted in an increased intracellular accumulation of T15-Id sIgM in the wild-type transfectant, but not in the B186 variant. Subcellular fractionation analysis revealed that the ligands bound to the T15-Id sIgM are not efficiently transported to the dense lysosomal compartments in both B186 and TM2 transfectants, as compared to the wild-type sIgM transfectant. A significant increase in tyrosine phosphorylation after cross-linking of the T15-Id sIgM was observed only in the wild-type sIgM transfectant. These results suggest that, while the NH2-terminal third of the TM region is not involved in the process responsible for the ligand-induced reduction of surface expression of sIgM, it appears to be essential for subsequent transport of sIgM/ligand complexes to the lysosomal compartments, as well as efficient activation of tyrosine kinases. These results strongly suggest that sIg-mediated enhancement of specific antigen presentation reflects the ability of sIg to efficiently transport antigen to the lysosomal compartments, and possibly the activation of protein tyrosine kinases.

Amino Acid Sequence↗
PubMed1994

Functional analysis of the V gamma 3 promoter of the murine gamma delta T-cell receptor.

The initial day 14 wave of fetal thymocytes express a gamma delta T-cell receptor (TCR). This surface TCR is generated by preferential rearrangement of V gamma 3 and V delta 1 recombination segments. To delineate the role of regulatory sequences in this expression, we have analyzed the V gamma 3 promoter control region under the regulation of its cognate C gamma 1 enhancer. Transcription initiates 25 bases downstream from a TATTAA sequence at a consensus initiator motif. The minimal 5' promoter sequences supporting expression by transient analysis extend -243 nucleotides from the +1 start site. Three regulatory sequences in this region have been defined by deletion and mutagenesis: a consensus CTF/NF-1 site at -55, an Ets homology sequence at -65, and a degenerate, but crucial, SP-1 site at -100. The presence of additional sequences downstream of the start site which extend through the leader intron were necessary for expression. In contrast to other TCR or immunoglobulin variable regions, one or more strong upstream suppressor sequences resembling silencer elements have been observed. A 311-bp fragment, positions -586 to -897, exhibited strong repressing activity regardless of orientation when placed upstream of heterologous promoters.

Animals↗
PubMed1993

DNA-binding properties and secondary structural model of the hepatocyte nuclear factor 3/fork head domain.

An 84-amino acid segment of QRF-1 [glutamine (Q)-rich factor 1], a newly cloned, B-cell-derived DNA-binding protein, shows significant sequence homology with the DNA-binding domains of the hepatocyte nuclear factor 3/fork head family of proteins. Here we demonstrate that this 84-amino acid domain is necessary and sufficient for DNA binding. We also propose a secondary structural model for the domain. At the N-terminal portion of the model, a basic hook structure is followed by two amphipathic helices separated by a turn. Invariant amino acid residues within the two proposed helices form the hydrophobic cores. An aromatic kink and a third amphipathic helix comprise the center of the domain. At the C terminus, two variable-length loops flank a putative 7-amino acid helix followed by a short basic region.

Amino Acid Sequence↗