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R H Scheuermann

Publications and source records attributed to R H Scheuermann.

44 records · Page 3Linked to original sources
PubMed1991

Anti-IgM antibodies down modulate mu-enhancer activity and OTF2 levels in LPS-stimulated mouse splenic B-cells.

Stimulation of small, resting, splenic B cells with bacterial lipopolysaccharide (LPS) induces proliferation, differentiation to plasma cell formation, and the expression of immunoglobulin heavy chain (IgH). When this is combined with agents which crosslink surface Ig, differentiation and the induction of surface immunoglobulin are suppressed even though proliferation proceeds. We find that anti-mu antibodies suppresses Ig gene expression of transfected mu constructs, even if either the membrane or secretory segments have been deleted. We examined the effects of anti-mu treatment on the IgH enhancer (IgHE) attached to a heterologous test gene (CAT). Indeed the IgH enhancer alone was subject to anti-mu suppression, while the SV40 enhancer was insensitive. To determine what was responsible for suppression of enhancer function by anti-mu we examined nuclear extracts from stimulated splenic B cells for the presence of sequence-specific DNA binding activities to various sites within the enhancer. We found two specific differences--an induction in mu E5 binding activity, and a reduction in octamer transcription factor 2 (OTF2) binding activity, after anti-mu treatment. Analysis of these cells by in situ immunofluorescence with anti-OTF2 antibodies suggests that the nuclear localization of OTF2 in anti-mu treated cells may change, as well as its absolute level.

Animals↗
PubMed1989

A developmental-specific factor binds to suppressor sites flanking the immunoglobulin heavy-chain enhancer.

We identified a novel nuclear protein, NF-mu NR, that binds to multiple sites flanking the immunoglobulin heavy-chain enhancer. The expression of NF-mu NR shows a unique developmental pattern; the activity is present in all cells representing early stages of B-cell development, but is absent from more mature cells that express a high level of immunoglobulin heavy chains. NF-mu NR also is present in most cell lines outside of the B-cell lineage (e.g., T cells, macrophages, and fibroblasts). The binding sites for NF-mu NR correlate very well with cis-acting negative regulatory elements of the heavy-chain enhancer defined previously. Indeed, when the segments bound by NF-mu NR are deleted from the enhancer, it is now found to function as a positive transcription element in T cells and macrophages. Taken together, these results suggest that NF-mu NR may function as a negative regulator of enhancer function. The observation that the segments bound by NF-mu NR correspond to the segments bound to the nuclear matrix suggests an intriguing model not only of how enhancers might function but also of how negative regulation might occur.

Animals↗
PubMed1988

UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA.

The mutation rate of Escherichia coli increases approximately 100-fold after treatment with replication-inhibiting agents such as UV light. This enhanced mutation rate requires the action of the UmuD and UmuC proteins, which are induced as part of the SOS response to DNA damage. To initiate a biochemical characterization of the role of these proteins, we have developed a plasmid system that gives efficient expression of the umuD and umuC genes. The umuD and umuC genes were placed under the control of a regulated phage lambda PL promoter and a synthetic ribosome-binding site, and the distance to the UmuD start was adjusted to maximize gene expression. Starting from this overproduction system, we have purified the UmuD protein and studied its interaction with RecA. The SOS response is turned on by the capacity of RecA protein to mediate cleavage of the LexA repressor for SOS-controlled operons. Others have shown that UmuD exhibits sequence homology to LexA around the cleavage site, suggesting a possible cleavage reaction for UmuD. We show that RecA mediates cleavage of UmuD, probably at this site. As with LexA, UmuD also undergoes a self-cleavage reaction. We infer that RecA-mediated cleavage of UmuD is another role for RecA in SOS mutagenesis, probably activating UmuD for its mutagenic function.

Bacterial Proteins↗
PubMed1986

Capacity of RecA protein to bind preferentially to UV lesions and inhibit the editing subunit (epsilon) of DNA polymerase III: a possible mechanism for SOS-induced targeted mutagenesis.

The RecA protein of Escherichia coli is required for SOS-induced mutagenesis in addition to its recombinational and regulatory roles. Most SOS-induced mutations probably occur during replication across a DNA lesion (targeted mutagenesis). We have suggested previously that RecA might participate in targeted mutagenesis by binding preferentially to the site of the DNA damage (e.g., pyrimidine dimer) because of its partially unwound character; DNA polymerase III (polIII) will then encounter RecA-coated DNA at the lesion and might replicate across the damaged site with reduced fidelity. In this report, we analyze at a biochemical level two major predictions of this model. With respect to lesion recognition, we show that purified RecA protein binds more efficiently to UV-irradiated double-stranded DNA than to nonirradiated DNA, as judged by filter-binding and gel electrophoresis assays. With respect to replication fidelity, Fersht and Knill-Jones [Fersht, A. R. & Knill-Jones, J. W. (1983) J. Mol. Biol. 165, 669-682] have found that RecA inhibits the 3'----5' exonuclease (editing function) of polIII holoenzyme. We extend this observation by demonstrating that RecA inhibits the exonuclease of the purified editing subunit of polIII, epsilon protein. Thus, we suggest that the activities of RecA required for targeted mutagenesis are lesion-recognition, followed by localized inhibition of the editing capacity of the epsilon subunit of polIII holoenzme. In this proposed mechanism, one activation signal for RecA for mutagenesis is the lesion itself. Because UV-irradiated, double-stranded DNA efficiently activates RecA for cleavage of the LexA repressor, the lesion itself may also often serve as an activation signal for induction of SOS-controlled genes.

Antibody Diversity↗
PubMed1984

A separate editing exonuclease for DNA replication: the epsilon subunit of Escherichia coli DNA polymerase III holoenzyme.

DNA polymerase III (polIII) holoenzyme of Escherichia coli has 3'----5' exonuclease ("editing") activity in addition to its polymerase activity, a property shared by other prokaryotic DNA polymerases. The polymerization activity is carried by the large alpha subunit, the product of the dnaE gene. Mutations affecting the fidelity of DNA replication in vivo and the activity of 3'----5' exonuclease assayed in vitro are found in the dnaQ gene, which specifies the epsilon subunit. To determine whether epsilon carries the 3'----5' exonuclease activity, we have used an overproduction protocol to purify epsilon separately from the other subunits of polIII holoenzyme. We find that epsilon has 3'----5' exonuclease activity indistinguishable from that of polIII core, the subassembly of polIII holoenzyme consisting of the alpha, epsilon, and theta subunits. We conclude that the editing and polymerization activities of polIII holoenzyme reside on distinct subunits, in contrast to DNA polymerase I of E. coli and DNA polymerase of phage T4. This functional separation may provide for regulation of exonucleolytic editing independently of polymerization, allowing cellular control of replication fidelity.

DNA Polymerase III↗
PubMedDate not supplied

Bcl-2 expression in Langerhans' cell histiocytosis.

Langerhans' cell histiocytosis (LCH) is an abnormal accumulation of dendritic histiocytes of unknown pathogenesis. It has recently been shown to be a clonal process. Bcl-2 is a proto-oncogene whose protein product is known to inhibit apoptosis. The overexpression of bcl-2 has been demonstrated in a number of neoplasms, presumably prolonging the survival of the neoplastic cells. We examined the expression of bcl-2 in normal Langerhans' cells in the skin and in LCH by immunohistochemistry for protein and in situ hybridization for mRNA to see if it could be implicated in the pathogenesis of this disorder. Additionally, we performed Southern analysis to determine if genomic rearrangement of the bcl-2 gene occurs in cases of LCH. Bcl-2 was not detected in normal skin Langerhans' cells. Eleven of thirteen cases of LCH demonstrated bcl-2 protein expression in the cytoplasm of the Langerhans' cells by immunohistochemistry, while 12 of 13 cases had evidence of bcl-2 mRNA by in situ hybridization. Southern analysis revealed a germline configuration of the bcl-2 gene in the five cases studied. These findings suggest that bcl-2 expression is present and up-regulated in pathologic Langerhans' cells, however, this overexpression does not appear to be due to genomic rearrangement.

Biomarkers↗
PubMedDate not supplied

Epstein-Barr virus polymerase chain reaction and serology in pediatric post-transplant lymphoproliferative disorder: three-year experience.

To assess whether the semiquantitative peripheral blood Epstein-Barr virus (EBV) polymerase chain reaction (PCR) test correlates with post-transplant lymphoproliferative disorder (LPD), we compiled the results of the test done over a 3-year period ending July 1997. Six hundred seventy-six tests were done on 185 patients. Four hundred-thirty tests (63%) were negative, 167 (25%) were weak positive, 67 (10%) were moderate positive, and 12 (2%) were strong positive. Twelve of the patients developed a lymphoproliferative disorder (LPD) during this time. The EBV PCR tests proximate to the diagnosis of LPD in the 12 patients with EBV-positive LPD were 6 strong positive, 5 moderate positive, 1 weak positive. No patient with LPD had a negative result at diagnosis. Stated another way, 6/12 (50%) of strong-positive PCR tests, 5/67 (7%) moderate-positive tests, and 1/167 (.6%) of weak-positive tests correlated with LPD. Serologic evaluation for EBV done on 7 patients at the time of LPD showed low serologic responses in 5 of the 7 patients. The EBV PCR temporally associated with the serology indicated moderate to large viral burdens. In each patient evaluated serially, the EBV PCR test rose before the diagnosis of LPD and fell with treatment for the disorder. In conclusion, the EBV PCR test may be used as an adjunct to the diagnosis of patients with LPD and may be used to monitor response to therapy for the disorder.

Antibodies, Viral↗