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Biomedical subjects

W Schaffner

Publications and source records attributed to W Schaffner.

At least 91 records · Page 5Linked to original sources
PubMed1996

Transcriptional repression by RING finger protein TIF1 beta that interacts with the KRAB repressor domain of KOX1.

Many of the vertebrate zinc finger factors of the Kruppel type (C2H2 zinc fingers) contain in their N-terminus a conserved sequence referred to as the KRAB (Kruppel-associated box) domain that, when tethered to DNA, efficiently represses transcription. Using the yeast two-hybrid system, we have isolated an 835 amino acid RING finger (C3HC4 zinc finger) protein, TIF1 beta (also named KAP-1), that specifically interacts with the KRAB domain of the human zinc finger factor KOX1/ZNF10. TIF1 beta, TIF1 alpha, PML and efp belong to a characteristic subgroup of RING finger proteins that contain one or two other Cys/His-rich clusters (B boxes) and a putative coiled-coil in addition to the classical C3HC4 RING finger motif (RBCC configuration). Like TIF1 alpha, TIF1 beta also contains an additional Cys/His cluster (PHD finger) and a bromo-related domain. When tethered to DNA, TIF1 beta can repress transcription in transiently transfected mammalian cells both from promoter-proximal and remote (enhancer) positions, similarly to the KRAB domain itself. We propose that TIF1 beta is a mediator of the transcriptional repression exerted by the KRAB domain.

Amino Acid Sequence↗
PubMed1996

Management of an outbreak of tuberculosis in a small community.

OBJECTIVE: To investigate an outbreak of tuberculosis, determine the number of active cases and infections, and examine efforts to control the spread of disease. SETTING: A small town in Maine, in which no cases of tuberculosis had been reported in the previous 3 years. DESIGN: Epidemiologic investigation of an outbreak of tuberculosis infection and disease. MEASUREMENTS: A patient with an active case of tuberculosis was defined as a resident of the town or the surrounding area or an employee of the local shipyard who had a culture of sputum or tissue that was positive for Mycobacterium tuberculosis between June 1989 and May 1992. A case of tuberculous infection was defined as a positive tuberculin skin test result in a person with no previous positive test result. RESULTS: 21 active cases of tuberculosis occurred among shipyard workers and persons residing in the affected community between 1989 and 1992. One patient was the source of the outbreak; 8 months lapsed between the onset of this patient's illness and appropriate diagnosis and treatment. The M. tuberculosis strains isolated from this patient and from six other patients belonged to phage type I, auxiliary 14. All isolates were susceptible to drug treatment. Of 9898 persons who were tested, 697 (7%) were newly infected. Because isoniazid prophylaxis was not routinely offered to infected persons older than 35 years of age, only 341 (49%) infected persons completed isoniazid prophylaxis. CONCLUSIONS: Many secondary cases of tuberculosis occurred throughout this small Maine community because of delayed diagnosis and treatment of the source patient, delayed outbreak investigation, and failure to promote isoniazid prophylaxis to all persons infected during the outbreak. Aggressive efforts to identify persons with new infection are of limited value in controlling tuberculosis unless they are accompanied by an equally aggressive use of isoniazid prophylaxis.

Adult↗
PubMed1996

The B cell coactivator Bob1 shows DNA sequence-dependent complex formation with Oct-1/Oct-2 factors, leading to differential promoter activation.

We have shown previously that both octamer binding transcription factors, namely the ubiquitous Oct-1 and the B cell-specific Oct-2A protein, can be enhanced in transcriptional activity by their association with the B cell-specific coactivator protein Bob1, also called OBF-1 or OCA-B. Here we study the structural requirements for ternary complex formation of DNA-Oct-Bob1 and coactivation function of Bob1. In analogy to DNA-bound transcription factors, Bob1 has a modular structure that includes an interaction domain (amino acids 1-65) and a C-terminal domain (amino acids 65-256), both important for transcriptional activation. A mutational analysis has resolved a region of seven amino acids (amino acids 26-32) in the N-terminus of Bob1 that are important for contacting the DNA binding POU domain of Oct-1 or Oct-2. In contrast to the viral coactivator VP16 (vmw65), which interacts with Oct-1 via the POU homeosubdomain, Bob1 association with Oct factors requires residues located in the POU-specific subdomain. Because the same residues are also involved in DNA recognition, we surmised that this association would affect the DNA binding specificity of the Oct-Bob1 complex compared with free Oct factors. While Oct-1 or Oct-2 bind to a large variety of octamer sequences, Bob1 ternary complex formation is indeed highly selective and occurs only in a subset of these sequences, leading to the differential coactivation of octamer-containing promoters. The results uncover a new level in selectivity that furthers our understanding in the regulation of cell type-specific gene expression.

Amino Acid Sequence↗
PubMed1996

Two versatile eukaryotic vectors permitting epitope tagging, radiolabelling and nuclear localisation of expressed proteins.

Two versatile eukaryotic expression vectors have been developed which permit the production of an epitope-tagged cDNA insert by transient transfection in mammalian cells or by in vitro transcription-translation. The first vector, pCATCH, can be used to clone cDNA inserts in three different frames via eight unique restriction sites in a multiple cloning site (MCS) located downstream from both the FLAG epitope and the specific heart muscle kinase phosphorylation site, conferring the possibility of in vitro radiolabelling. A specific protease cleavage site enables the removal of the FLAG epitope, simplifying affinity purification of recombinant CATCH proteins. pCATCH possesses stop codons in all three reading frames at the 3' terminal end of the MCS. A derivate of this vector, pCATCH-NLS, was constructed by incorporating an SV40 nuclear localisation signal upstream from the MCS, for directed localisation of the tagged proteins.

Amino Acid Sequence↗
PubMed1996

Transcriptional repression by methylation: cooperativity between a CpG cluster in the promoter and remote CpG-rich regions.

Cytosine methylation of binding sites for transcription factors is a straightforward mechanism to prevent transcription, while data on an indirect mechanism, by methylation outside of the factor binding sites, are still scarce. We have studied the latter effect using a model promoter construct. For this, a 69 bp G + C rich DNA segment with a cluster of 14 CpG sites was inserted between upstream lexA sites and the TATA box. Transcription was measured in transient transfection assays with lexA-VP16 as an activating factor. When the entire plasmid was methylated at all CpGs before transfection, transcription was blocked (to 3% residual activity), whereas transcription was only mildly inhibited (to 60%) by methylation of a control plasmid that lacked the 69 bp CpG cluster. However, the effect could not simply be attributed to methylation of the CpG cluster: neither a methylated CpG cluster in an otherwise methylation-free reporter gene plasmid, nor the methylated plasmid with an unmethylated CpG cluster, inhibited transcription considerably (69% and 44% remaining activity, respectively). The data presented here suggest that a minimal length of methylated DNA in the promoter is required for repression, and imply that concomitant methylation of CpGs in the promoter region and in remote sequences can cooperatively block transcription, without the need to methylate any binding sites for transcription factors. We also note that the cooperation for a negative effect described here bears an analogy to transcriptional activation, where a promoter often cooperates with a remote enhancer.

Base Sequence↗
PubMed1996

Adult immunizations.

New vaccines have been licensed for hepatitis A, varicella, and typhoid. This paper reviews these vaccines and their recommended uses in adults. Special attention is given to a new national policy establishing age 50 years as a time for review of preventive health measures with emphasis on evaluating risk factors that indicate a need for pneumococcal vaccine and the initiation of annual influenza immunization.

Adult↗
PubMed1996

B lymphocytes are impaired in mice lacking the transcriptional co-activator Bob1/OCA-B/OBF1.

The protein Bob1 (also called OCA-B or OBF-1) is a B lymphocyte-specific transcriptional co-activator which can stimulate transcription by interaction with Oct-1 or Oct-2 and the octamer sequence found in the promoter and most enhancer regions of the immunoglobulin genes. The role that Bob1 plays in the maturation and function of B cells was investigated in mice deficient for Bob1. Although early stages of B cell development in the bone marrow do not appear to be affected, these mice show reduced numbers of B cells in peripheral lymphoid organs. Based on staining for B220, heat-stable antigen, and IgD, this reduction is particularly strong in those cells representing the more mature B cell stages. Probably as a consequence of this reduction in mature B cells, Bob1-deficient mice show reduced serum titers of the immunoglobulin isotypes IgG1, IgG2a, IgG2b and IgA, but not IgM. Histological examination of sections from spleen and lymph nodes reveal that while Bob-1-deficient mice have primary follicles, they lack well-developed germinal centers. Interestingly, B1 (Ly1) B cells in the peritoneum do not appear to be affected by the lack of Bob1. Taken together, these results suggest that, at least in conventional B cells, Bob1 plays in important role in the antigen-driven stages of B cell activation and maturation.

Animals↗
PubMed1996

Differential sensitivity of zinc finger transcription factors MTF-1, Sp1 and Krox-20 to CpG methylation of their binding sites.

Cytosine methylation at CpG sites is often negatively correlated with mammalian gene activity. Many transcription factors whose DNA binding site contains one or more CpG dinucleotides are no longer able to efficiently bind DNA when the site is methylated. A notable exception is the zinc finger factor Sp1 which binds DNA and activates transcription even when its binding site is methylated. Here we show that two other zinc finger factors, MTF-1 and Krox-20, can also bind to CpG methylated sites. MTF-1 regulates metallothionein gene transcription by binding to a number of metal responsive elements (MREs), and Krox-20 regulates Hox genes during hindbrain segmentation. However, a refined analysis of MTF-1/MRE binding shows that methylation is not tolerated at every binding site: the highest affinity site in the mouse metallothionein I gene, MREd, is unaffected by methylation, while two other MRE sites with CpGs at different positions are rendered partially or completely nonfunctional by methylation. Both methylation sensitive and insensitive factors/binding sites are likely to determine the developmental expression pattern of a gene.

Animals↗
PubMed1996

Cloning and characterization of the murine B-cell specific transcriptional coactivator Bob1.

From a murine B-cell cDNA-library we have cloned a cDNA encoding the murine B-cell specific coactivator mBob1. The protein is the murine homologue to the recently described human coactivator Bob1 (hBob1), also referred to as OBF-1 or OCA-B. We have also characterized the genomic mBob1 clone. Analysis of its intron-exon structure has allowed identification of a C-terminal splice variant. mBob1 is B-cell restricted, and is found in all B-cell lines representing different stages of B-cell differentiation. mBob1 interacts with the octamer transcription factors Oct-1 and Oct-2 and stimulates transcription mediated by these factors.

Amino Acid Sequence↗
PubMed1996

Dramatic changes in the ratio of homologous recombination to nonhomologous DNA-end joining in oocytes and early embryos of Xenopus laevis.

We have developed a versatile plasmid vector (pReco-sigma) for recombination studies. When linearized and introduced into the cells of interest, pReco-sigma allows the simultaneous determination of the relative frequencies of homologous recombination versus nonhomologous DNA-end joining (also termed end-to-end joining), the latter an example of illegitimate recombination processes. As a system we made use of stage VI oocytes and fertilized eggs of the African clawed frog Xenopus laevis, which were previously described to support homologous recombination and DNA-end joining, respectively. Extending these earlier findings, we show that oocytes yield > 80% of the homologously recombined product, whereas in eggs a highly efficient DNA-end joining activity predominates (> 95%). Both reactions, homologous recombination and DNA-end joining, are shown to occur quickly, with the majority of the respective products being formed within the first 20 minutes of incubation under optimal conditions. In fertilized eggs, up to 50% of all injected linear DNA molecules are recircularized by DNA-end joining. With high amounts of injected DNA per fertilized egg, DNA-end joining is reduced, presumably due to competition for essential factors, and homologous recombination becomes readily detectable. As there is a sequence of rapid cleavage divisions after fertilization of the egg, the fast and highly efficient DNA-end joining, even though it is error-prone at the junction site, seems to be best suited to cope with DNA double-strand breaks that might occur in the genome during early embryogenesis. On the other hand, the long-lived oocytes seem to repair DNA double-strand breaks via homologous recombination. This latter property may be exploited both in Xenopus and in other organisms to achieve homologous integration of exogenous DNA into germ cells for gene targeting.

Animals↗
PubMed1996

Improved "activator trap" method for the isolation of transcriptional activation domains from random DNA fragments.

We have previously developed an "activator trap" method for selective isolation of activation domains from viral and cellular transcription factors. In this method, random sonicated DNA fragments were ligated next to the DNA binding domain (DBD) of the GAL4 factor in a plasmid that also contained a simian virus 40 (SV40) replication origin. A library of such random insert plasmids was transfected into a monkey cell line (CV-1-5GT), which had been stably transformed with a GAL4-inducible SV40 T-antigen gene. Chimeric GAL factors with a heterologous activation domain were harvested after selective replication in these CV-1-5GT cells. Here we report a simplification and generalization of the "activator trap" method. First, the time-consuming library construction step can be omitted by direct transfection of the sonicated DNA fragments and the linearized recipient plasmid vector into CV-1-5GT cells to obtain chimeric GAL-activation factors by in vivo ligations. Second, the dependence on CV-1-5GT cells can be bypassed by direct co-transfection of all components, including a plasmid carrying the T-antigen gene into cells other than CV-1-5GT. This latter step allows the application of the method to cultured human cells, as demonstrated with the human B-cell line BJA-B.

Animals↗
PubMed1996

BZLF1 (ZEBRA, Zta) protein of Epstein-Barr virus selected in a yeast one-hybrid system by binding to a consensus site in the IgH intronic enhancer: a role in immunoglobulin expression?

We have used a yeast one hybrid screen to search for factors interacting with a subsegment of the immunoglobulin heavy chain (IgH) intronic enhancer. The 51 bp enhancer segment harbored a so-called E-box and an octamer site, known to bind helix-loop-helix transcription factors and Oct factors, respectively. Mammalian Oct-2A protein was also expressed in yeast, to select for transcription factors possibly cooperating with Oct-2. Six strongly interacting protein clones were selected from a peripheral blood lymphocyte library. These included a B cell-specific co-activator, termed Bob1, that directly binds to Oct-2 (Gstaiger et al., 1995, Nature 373, 360-362). Three further clones represent the helix-loop factors ITF-1 and ITF-2, another one the nucleolar protein nucleophosmin, or B23. Unexpectedly, the sixth clone with strong activity encoded the BZLF1 (= ZLF1, zta, ZEBRA, EB1) protein of Epstein-Barr virus (EBV). BZLF1 is a leucine zipper-related transcription factor and induces the switch from viral latency to lytic growth. We found that BZLF1 also activated transcription in transiently transfected mammalian cells via a consensus binding site located within the IgH intron enhancer. BZLF1 may thus influence immunoglobulin heavy chain expression in EBV-infected B lymphocytes.

Consensus Sequence↗
PubMed1995

Transcription factors interacting with herpes simplex virus alpha gene promoters in sensory neurons.

Interference with VP16-mediated activation of herpes virus immediate-early (or alpha) genes is thought to be the major cause of establishing viral latency in sensory neurons. This could be brought about by lack of a key activating transcription factor(s) or active repression. In this study we find that sensory neurons express all important components for VP16-mediated alpha gene induction, such as the POU transcription factor Oct-1, host cell factor (HCF) and GABP alpha/beta. However, Oct-1 and GABP alpha/beta are only present at low levels and the VP16-induced complex (VIC) appears different. We do not find protein expression of the transcription factor Oct-2, implicated by others as an alpha gene repressor. The POU factor N-Oct3 (Brn 2 or POU3F2) is also present in sensory neurons and binds viral TAATGARAT motifs with higher affinity than Oct-1, indicating that it may be a candidate repressor for competitive binding to TAATGARAT motifs. When transfected into HeLa cells, where Oct-1 and GABP alpha/beta are highly abundant, N-Oct3 represses model promoters with multimerized TAATGARAT motifs, but fails to repress complete alpha gene promoters. Taken together our findings suggest that modulation of alpha gene promoters could contribute to viral latency when low concentrations of the activating transcription factors Oct-1 and GABP alpha/beta prevail. Our data, however, refute the notion that competing Oct factors are able to block alpha gene transcription to achieve viral latency.

Animals↗