The Wilms' tumor locus (WTL) at 11p13 contains a gene that encodes a zinc finger-containing protein that has characteristics of a DNA-binding protein. However, binding of this protein to DNA in a sequence-specific manner has not been demonstrated. A synthetic gene was constructed that contained the zinc finger region, and the protein was expressed in Escherichia coli. The recombinant protein was used to identify a specific DNA binding site from a pool of degenerate oligonucleotides. The binding sites obtained were similar to the sequence recognized by the early growth response-1 (EGR-1) gene product, a zinc finger-containing protein that is induced by mitogenic stimuli. A mutation in the zinc finger region of the protein originally identified in a Wilms' tumor patient abolished its DNA-binding activity. These results suggest that the WTL protein may act at the DNA binding site of a growth factor-inducible gene and that loss of DNA-binding activity contributes to the tumorigenic process.

The wt1 gene, a putative tumor suppressor gene located at the Wilms tumor (WT) locus on chromosome 11p13, encodes a zinc finger-containing protein that binds to the same DNA sequence as EGR-1, a mitogen-inducible immediate-early gene product that activates transcription. The transcriptional regulatory potential of WT1 has not been demonstrated. In transient transfection assays, the WT1 protein functioned as a repressor of transcription when bound to the EGR-1 site. The repression function was mapped to the glutamine- and proline-rich NH2-terminus of WT1; fusion of this domain to the zinc finger region of EGR-1 converted EGR-1 into a transcriptional repressor.

WTI is a tumor-suppressor gene expressed in the developing kidney, whose inactivation leads to the development of Wilms tumor, a pediatric kidney cancer. WTI encodes a transcription factor which binds to the EGRI consensus sequence, mediating transcriptional repression. We now demonstrate that p53, the product of a tumor-suppressor gene with ubiquitous expression, physically associates with WT1 in transfected cells. (2,6). WTI mutations detected in Wilms tumor specimens have also provided reagents to dissect the functional properties of WT1 protein.Of particular interest is a dominant negative mutation, WTAR (7), which encodes an in-frame deletion of the third zinc finger and demonstrates oncogenic potential in baby rat kidney (BRK) cell transformation assays (8). Disruption of the DNA-binding domain by the WTAR mutation suggests that its dominant effect may result from interactions with other cellular proteins.Using stable BRK cell lines immortalized by transfection with the adenovirus EIA gene along with WTI, we demonstrate the presence of a complex containing WT1 and p53 proteins. This complex is also observed in BRK cells expressing mutant WTAR and a mutated p53 gene (codon 248), as well as in specimens of sporadic Wilms tumor. A potential functional interaction between WT1 and p53 is suggested by transactivation assays using their respective target sequences. While WT1 enhances transcriptional activation by p53, wild-type p53 appears to convert WT1 from a transcriptional activator to a transcriptional repressor. MATERIALS AND METHODSImmunoprecipitations and Western Blot Analyses. Three anti-WT1 antibodies were used: WT-6F1 and WT-91 areThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. rabbit polyclonal antibodies directed against overlapping N-terminal peptides, aa 1-173 and 85-173, respectively (9); DG10 is a monoclonal antibody generated against human WT1 synthesized in Escherichia coli. For immunoprecipitations, cells were labeled with [35S]methionine and extracted with either ELB buffer (50 mM Hepes, pH 7.0/250 mM NaCl/0.5 mM EDTA/0.1% Nonidet P-40) or RIPA buffer (10 mM Tris, pH 7.4/150 mM NaCl, 1% Triton X-100/1% sodium deoxycholate/0.1% SDS). For sequential immunoprecipitations, cellular lysates were extracted with ELB buffer and immunoprecipitated with the first antibody. The immune complex was then dissociated in RIPA buffer and the released proteins were immunoprecipitated with the second antibody. Peptide maps of immunoprecipitated WT1 proteins were generated using various concentrations of Staphylococcus aureus V8 protease (10). For immunoprecipitation/ Western analysis, anti-p53 antibodies were covalently crosslinked to protein A-Sepharose (10), and after immunoprecipitation, proteins were released from the antibody by incubation with 2% SDS/50 mM Tris, pH 6.8, at room temperature for 5 min. Western blot analysis was performed a...

The myeloid zinc finger gene 1, MZFI, encodes a transcription factor which is expressed in hematopoietic progenitor cells that are committed to myeloid lineage differentiation. MZF1 contains 13 C2H2 zinc fingers arranged in two domains which are separated by a short glycine-and proline-rich sequence. The first domain consists of zinc fingers 1 to 4, and the second domain is formed by zinc fingers 5 to 13. We have determined that both sets of zinc finger domains bind DNA. Purified, recombinant MZF1 proteins containing either the first set of zinc fingers or the second set were prepared and used to affinity select DNA sequences from a library of degenerate oligonucleotides by using successive rounds of gel shift followed by PCR amplification. Surprisingly, both DNA-binding domains of MZF1 selected similar DNA-binding consensus sequences containing a core of four or five guanine residues, reminiscent of an NF-KB half-site: 1-4, 5'-AGTGGGGA-3'; 5-13, 5'-CGGGnGAGGGGGAA-3'. The full-length MZF1 protein containing both sets of zinc finger DNAbinding domains recognizes synthetic oligonucleotides containing either the 1-4 or 5-13 consensus binding sites in gel shift assays. Thus, we have identified the core DNA consensus binding sites for each of the two DNA-binding domains of a myeloid-specific zinc finger transcription factor. Identification of these DNAbinding sites will allow us to identify target genes regulated by MZF1 and to assess the role of MZF1 as a

Change combines cutting-edge scientific research with independent policy analysis to provide a solid foundation for the public and private decisions needed to mitigate and adapt to unavoidable global environmental changes. Being data-driven, the Joint Program uses extensive Earth system and economic data and models to produce quantitative analysis and predictions of the risks of climate change and the challenges of limiting human influence on the environmentessential knowledge for the international dialogue toward a global response to climate change.To this end, the Joint Program brings together an interdisciplinary group from two established MIT research centers: the Center for Global Change Science (CGCS) and the Center for Energy and Environmental Policy Research (CEEPR). These two centers-along with collaborators from the Marine Biology Laboratory (MBL) at Woods Hole and short-and long-term visitors-provide the united vision needed to solve global challenges.At the heart of much of the program's work lies MIT's Integrated Global System Model. Through this integrated model, the program seeks to discover new interactions among natural and human climate system components; objectively assess uncertainty in economic and climate projections; critically and quantitatively analyze environmental management and policy proposals; understand complex connections among the many forces that will shape our future; and improve methods to model, monitor and verify greenhouse gas emissions and climatic impacts.This reprint is intended to communicate research results and improve public understanding of global environment and energy challenges, thereby contributing to informed debate about climate change and the economic and social implications of policy alternatives. Given uncertainty in long-term carbon reduction goals, how much non-carbon generation should be developed in the near-term? This research investigates the optimal balance between the risk of overinvesting in non-carbon sources that are ultimately not needed and the risk of underinvesting in non-carbon sources and subsequently needing to reduce carbon emissions dramatically. We employ a novel framework that incorporates a computable general equilibrium (CGE) model of the U.S. into a two-stage stochastic approximate dynamic program (ADP) focused on decisions in the electric power sector. We solve the model using an ADP algorithm that is computationally tractable while exploring the decisions and sampling the uncertain carbon limits from continuous distributions.The results of the model demonstrate that an optimal hedge is in the direction of more non-carbon investment in the near-term, in the range of 20-30% of new generation. We also demonstrate that the optimal share of non-carbon generation is increasing in the variance of the uncertainty about the long-term carbon targets, and that with greater uncertainty in the future policy regime, a balanced portfolio of non-carbon, natural gas, and coal generation is desirable.

SummaryCostimulation mediated by the CD28 molecule plays an important role in optimal activation of T cells. However, CD28-deficient mice can mount effective T cell-dependent immune responses, suggesting the existence of other costimulatory systems. In a search for other costimulatory molecules on T cells, we have developed a monoclonal antibody (mAb) that can costimulate T cells in the absence of antigen-presenting cells (APC). The molecule recognized by this mAb, 9D3, was found to be expressed on almost all mature T cells and to be a protein of ,'-~24 kD molecular mass. By expression cloning, this molecule was identified as CD9. 9D3 (anti-CD9) synergized with suboptimal doses of anti-CD3 mAb in inducing proliferation by virgin T cells. Costimulation was induced by independent ligation of CD3 and CD9, suggesting that colocalization of these two molecules is not required for T cell activation. The costimulation by anti-CD9 was as potent as that by anti-CD28. Moreover, anti-CD9 costimulated in a CD28-independent way because anti-CD9 equally costimulated T cells from the CD28-deficient as well as wild-type mice. Thus, these results indicate that CD9 serves as a molecule on T cells that can deliver a potent CD28-independent costimulatory signal. Full activation of the T cell has been shown to require two independent signals (1). The first signal is provided by antigen-specific T cell receptor (TCR) interacting with processed antigen peptides plus major histocompatibitity complex (MHC) molecules on APC. This signal leads to an effective T cell response only when accompanied by a second costimulatory signal(s) presented by the APC. The lack of costimulation not only prevents activation but also induces tolerance called anergy (1). Identifying molecules capable of delivering costimulatory signals has been the subject of a large number of recent investigations (2-5). CD28 expressed on T cells was found to be a receptor for the costimulatory molecules CD80 and CD86 on APC (6). CD28 engagement, by either anti-CD28 mAb or ligands (CD80/ CD86), has been shown to costimulate T cells in the absence of APC, resulting in T cell activation (7-9). Conversely, the blocking of CD28-1igand interactions induced substantial inhibition of T cell activation (2). These observations indicated that the CD28-CD80/CD86 interaction functions as a critical pathway of T cell costimulation. Nevertheless, recent studies have revealed that CD28-deficient mice can develop normal in vivo immune responses (10) and that T cells from these mice mount APC-dependent responses for T cell activa~:ion in vitro although the response is reduced compared to T cells from wild-type mice (10, 11). Thus, these results strongly suggest that there may exist other molecules capable of providing costimulatory activity.In this report, we have developed a rat IgG mAb (9D3) by immunization with cells of a murine thymic stromal clone (12). This mAb recognized a protein of"-,24 kD that is expressed on immunizing thymic stromal cells as well as murine T cells. By cDNA expr...

Here we review our current results studying B cells as APC and the mechanisms by which processed antigen is transported to and held on the cell surface for recognition by the specific T cell along with the MHC class II molecules. These studies were carried out using the globular protein cytochrome c as antigen for which the T-cell antigenic determinant was localized to a C-terminal 10-amino acid peptide fragment. For certain analyses, native cytochrome c or antigenic peptide fragments were covalently coupled to antibodies directed toward B-cell surface structures, allowing the targeting of antigen to the APC surface. Our findings indicate that all B cells function as APC and that the APC function is not differentially regulated in defined B-cell subpopulations. Using cytochrome c-antibody conjugates, it was shown that the surface Ig plays two significant roles in augmenting the B-cell APC function following antigen binding: signalling for enhanced APC function and concentrating antigen for subsequent internalization and processing. Both IgM and IgD appear to function identically in facilitating antigen processing in both immune and nonimmune B-cell populations. Furthermore, the surface Ig does not appear to be specially differentiated to function in concentrating antigen, as antigen artificially bound to other B-cell surface structures including MHC class I and class II molecules is also effectively presented. Lastly, evidence is presented that a previously described B-cell activating factor activity is strongly associated with the membranes of activated but not unactivated helper T cells, providing a mechanism by which the T-cell helper function can be focused on the specific antigen-presenting B cell. Concerning the mechanism by which processed antigen is presented at the B-cell surface, evidence is presented suggesting a role of peptide-binding chaperone proteins which may function to transport peptide to the APC surface and facilitate its association with the appropriate Ia. One candidate protein, PBP72/74, is described which binds peptides but not native antigens, is a member of the hsp70 family and appears to play a role in antigen presentation by the ability of antisera raised against it to block APC functions. Peptide-antibody conjugates were used to explore the spacial restrictions on MHC-restricted peptide presentation and it was shown that peptides covalently coupled to antibodies specific for Ig, class I or class II molecules are effective antigens in vitro even in the absence of processing.(ABSTRACT TRUNCATED AT 400 WORDS)

Change combines cutting-edge scientific research with independent policy analysis to provide a solid foundation for the public and private decisions needed to mitigate and adapt to unavoidable global environmental changes. Being data-driven, the Joint Program uses extensive Earth system and economic data and models to produce quantitative analysis and predictions of the risks of climate change and the challenges of limiting human influence on the environmentessential knowledge for the international dialogue toward a global response to climate change.To this end, the Joint Program brings together an interdisciplinary group from two established MIT research centers: the Center for Global Change Science (CGCS) and the Center for Energy and Environmental Policy Research (CEEPR). These two centers-along with collaborators from the Marine Biology Laboratory (MBL) at Woods Hole and short-and long-term visitors-provide the united vision needed to solve global challenges.At the heart of much of the program's work lies MIT's Integrated Global System Model. Through this integrated model, the program seeks to discover new interactions among natural and human climate system components; objectively assess uncertainty in economic and climate projections; critically and quantitatively analyze environmental management and policy proposals; understand complex connections among the many forces that will shape our future; and improve methods to model, monitor and verify greenhouse gas emissions and climatic impacts.This reprint is intended to communicate research results and improve public understanding of global environment and energy challenges, thereby contributing to informed debate about climate change and the economic and social implications of policy alternatives. Given uncertainty in long-term carbon reduction goals, how much non-carbon generation should be developed in the near-term? This research investigates the optimal balance between the risk of overinvesting in non-carbon sources that are ultimately not needed and the risk of underinvesting in non-carbon sources and subsequently needing to reduce carbon emissions dramatically. We employ a novel framework that incorporates a computable general equilibrium (CGE) model of the U.S. into a two-stage stochastic approximate dynamic program (ADP) focused on decisions in the electric power sector. We solve the model using an ADP algorithm that is computationally tractable while exploring the decisions and sampling the uncertain carbon limits from continuous distributions.The results of the model demonstrate that an optimal hedge is in the direction of more non-carbon investment in the near-term, in the range of 20-30% of new generation. We also demonstrate that the optimal share of non-carbon generation is increasing in the variance of the uncertainty about the long-term carbon targets, and that with greater uncertainty in the future policy regime, a balanced portfolio of non-carbon, natural gas, and coal generation is desirable.