The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins.

Although transcription and pre-mRNA processing are colocalized in eukaryotic nuclei, molecules linking these processes have not previously been described. We have identified four novel rat proteins by their ability to interact with the repetitive C-terminal domain (CTD) of RNA polymerase II in a yeast two-hybrid assay. A yeast homolog of one of the rat proteins has also been shown to interact with the CTD. These CTD-binding proteins are all similar to the SR (serine/arginine-rich) family of proteins that have been shown to be involved in constitutive and regulated splicing. In addition to alternating Ser-Arg domains, these proteins each contain discrete N-terminal or C-terminal CTD-binding domains. We have identified SR-related proteins in a complex that can be immunoprecipitated from nuclear extracts with antibodies directed against RNA polymerase II. In addition, in vitro splicing is inhibited either by an antibody directed against the CTD or by wild-type but not mutant CTD peptides. Thus, these results suggest that the CTD and a set of CTD-binding proteins may act to physically and functionally link transcription and pre-mRNA processing.

Purification of the Drosophila RNA polymerase II general transcription factors.

We describe a fractionation and purification scheme for the Drosophila RNA polymerase II general transcription factors. Drosophila TFIIE, TFIIF, TFIIH, and RNA polymerase II have been purified to greater than 50% homogeneity from Drosophila embryo nuclear extracts. TFIID has been purified 80-fold and is not significantly contaminated with any of the other general factors. This is the first reported identification and purification of Drosophila TFIIH and TFIIE. Further analysis shows that, similar to their mammalian counterparts, Drosophila TFIIH is composed of eight polypeptides sized between 30 and 100 kDa, and Drosophila TFIIE is composed of two polypeptides sized at 34 and 60 kDa. When all of these fractions are combined with recombinant Drosophila TFlIB, a highly purified in vitro transcription system is generated that has not previously been available in Drosophila. The TFIID fraction can be replaced with recombinant Drosophila TBP to give a transcription system that is nearly free of contaminating proteins.

The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

Inhibition of the association of RNA polymerase II with the preinitiation complex by a viral transcriptional repressor.

Transcriptional repression is an important component of regulatory networks that govern gene expression. In this report, we have characterized the mechanisms by which the immediate early protein 2 (IE2 or IE86), a master transcriptional regulator of human cytomegalovirus, down-regulates its own expression. In vitro transcription and DNA binding experiments demonstrate that IE2 blocks specifically the association of RNA polymerase II with the preinitiation complex. Although, to our knowledge, this is the first report to describe a eukaryotic transcriptional repressor that selectively impedes RNA polymerase II recruitment, we present data that suggest that this type of repression might be widely used in the control of transcription by RNA polymerase II.

Quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II: a systematic study of the effect of carotenoid structure.

The role of carotenoids in quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II has been studied with a view to understanding the molecular basis of the control of photoprotective nonradiative energy dissipation by the xanthophyll cycle in vivo. The control of chlorophyll fluorescence quenching in the isolated complex has been investigated in terms of the number of the conjugated double bonds for a series of carotenoids ranging from n = 5-19, giving an estimated first excited singlet state energy from 20,700 cm-1 to 10,120 cm-1. At pH 7.8 the addition of exogenous carotenoids with >=10 conjugated double bonds (including zeaxanthin) stimulated fluorescence quenching relative to the control with no added carotenoid, whereas those with n the light-harvesting complex of photosystem II was induced by a lowering of pH to 5.5, carotenoids with n of fluorescence quenching relative to the control. Of the 10 carotenoids tested, quenching induced by the addition of the tertiary amine compound, dibucaine, to isolated light-harvesting complex of photosystem II could only be reversed by violaxanthin. These results are discussed in terms of the two theories developed to explain the role of zeaxanthin and violaxanthin in nonphotochemical quenching of chlorophyll fluorescence.

Truncation of the class II beta-chain cytoplasmic domain influences the level of class II/invariant chain-derived peptide complexes.

Previous studies have established that antigen presenting cells (APC) expressing major histocompatibility complex class II beta chains with truncated cytoplasmic domains are impaired in their capacity to activate T cells. While it had been widely accepted that this impairment is due to a defect in class II cytoplasmic domain-dependent signal transduction, we recently generated transgenic mice expressing only truncated class II beta chains, and functional analyses of APC from these mice revealed signaling-independent defects in antigen presentation. Here, we demonstrate that T cells primed on such transgenic APC respond better to stimulation by APC expressing truncated beta chains than by wild-type APC. This finding suggests that APC expressing truncated class II beta chains are not inherently defective in their antigen presenting capacity but, rather, may differ from wild-type APC in the peptide antigens that they present. Indeed, analysis of the peptides bound to class II molecules isolated from normal and transgenic spleen cells revealed clear differences. Most notably, the level of class II-associated invariant chain-derived peptides (CLIP) is significantly reduced in cells expressing only truncated beta chains. Prior studies have established that CLIP and antigenic peptides compete for binding to class II molecules. Thus, our results suggest that the cytoplasmic domain of the class II beta chain affects antigen presentation by influencing the level of CLIP/class II complexes.

The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer.

The type 1 angiotensin II (AT1) receptor is well characterized but the type 2 (AT2) receptor remains an enigma. We tested the hypothesis that the AT2 receptor can modulate the growth of vascular smooth muscle cells by transfecting an AT2 receptor expression vector into the balloon-injured rat carotid artery and observed that overexpression of the AT2 receptor attenuated neointimal formation. In cultured smooth muscle cells, AT2 receptor transfection reduced proliferation and inhibited mitogen-activated protein kinase activity. Furthermore, we demonstrated that the AT2 receptor mediated the developmentally regulated decrease in aortic DNA synthesis at the latter stages of gestation. These results suggest that the AT2 receptor exerts an antiproliferative effect, counteracting the growth action of AT1 receptor.

Proximity of the manganese cluster of photosystem II to the redox-active tyrosine YZ.

Electron spin echo electron-nuclear double resonance (ESE-ENDOR) experiments performed on a broad radical electron paramagnetic resonance (EPR) signal observed in photosystem II particles depleted of Ca2+ indicate that this signal arises from the redox-active tyrosine YZ. The tyrosine EPR signal width is increased relative to that observed in a manganese-depleted preparation due to a magnetic interaction between the photosystem II manganese cluster and the tyrosine radical. The manganese cluster is located asymmetrically with respect to the symmetry-related tyrosines YZ and YD. The distance between the YZ tyrosine and the manganese cluster is estimated to be approximately 4.5 A. Due to this close proximity of the Mn cluster and the redox-active tyrosine YZ, we propose that this tyrosine abstracts protons from substrate water bound to the Mn cluster.

Molecular characterization of PsbW, a nuclear-encoded component of the photosystem II reaction center complex in spinach.

We describe the isolation and characterization of cDNAs encoding the precursor polypeptide of the 6.1-kDa polypeptide associated with the reaction center core of the photosystem II complex from spinach. PsbW, the gene encoding this polypeptide, is present in a single copy per haploid genome. The mature polypeptide with 54 amino acid residues is characterized by a hydrophobic transmembrane segment, and, although an intrinsic membrane protein, it carries a bipartite transit peptide of 83 amino acid residues which directs the N terminus of the mature protein into the chloroplast lumen. Thylakoid integration of this polypeptide does not require a delta pH across the membrane, nor is it azide-sensitive, suggesting that the polypeptide chain inserts spontaneously in an as yet unknown way. The PsbW mRNA levels are light regulated. Similar to cytochrome b559 and PsbS, but different from the chlorophyll-complexing polypeptides D1, D2, CP43, and CP47 of photosystem II, PsbW is present in etiolated spinach seedlings.

Ectopic expression of the agouti gene in transgenic mice causes obesity, features of type II diabetes, and yellow fur.

Mice that carry the lethal yellow (Ay) or viable yellow (Avy) mutation, two dominant mutations of the agouti (a) gene in mouse chromosome 2, exhibit a phenotype that includes yellow fur, marked obesity, a form of type II diabetes associated with insulin resistance, and an increased susceptibility to tumor development. Molecular analyses of these and several other dominant "obese yellow" a-locus mutations suggested that ectopic expression of the normal agouti protein gives rise to this complex pleiotropic phenotype. We have now tested this hypothesis directly by generating transgenic mice that ectopically express an agouti cDNA clone encoding the normal agouti protein in all tissues examined. Transgenic mice of both sexes have yellow fur, become obese, and develop hyperinsulinemia. In addition, male transgenic mice develop hyperglycemia by 12-20 weeks of age. These results demonstrate conclusively that the ectopic agouti expression is responsible for most, if not all, of the phenotypic traits of the dominant, obese yellow mutants.

The glucocorticoid receptor type II complex is a target of the HIV-1 vpr gene product.

The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 15-kDa virion-associated protein that functions as a regulator of cellular processes linked to the HIV life cycle. We report the interaction of a 41-kDa cytosolic viral protein R interacting protein 1 (Rip-1) with Vpr in vitro. Rip-1 displays a wide tissue distribution, including relevant targets of HIV infection. Vpr protein induced nuclear translocation of Rip-1, as did glucocorticoid receptor (GR)-II-stimulating steroids. Importantly, Vpr and Rip-1 coimmunoprecipitated with the human GR as part of an activated receptor complex. Vpr complementation of a vpr mutant virus was also mimicked by GR-II-stimulating steroids. Vpr and GR-II actions were inhibited by mifepristone, a GR-II pathway inhibitor. Together these data directly link the activity of the vpr gene product to the glucocorticoid steroid pathway and provide a biochemical mechanism for the cellular and viral activity of Vpr, as well as suggest that a unique class of antivirals, which includes mifepristone (RU486), may influence HIV-1 replication.

Account book of Mary Hains as executrix of the estate of her husband, James Hains, 1784-1786

James and Mary Hains were residents of Mamaroneck, N.Y.

John Warren's copy of votes from a meeting of the Boston Medical Society, 1784 May 3

This document lists the eleven votes cast at a meeting of the Boston Medical Society on May 3, 1784. It was authorized as a "true coppy" by Thomas Kast, the Secretary of the Society. The following members of the Society were present at the meeting, all of them doctors: James Pecker, James Lloyd, Joseph Gardner, Samuel Danforth, Isaac Rand, Jr., Charles Jarvis, Thomas Kast, Benjamin Curtis, Thomas Welsh, Nathaniel Walker Appleton, and doctors whose last names were Adams, Townsend, Eustis, Homans, and Whitwell. The document indicates that a meeting had been held the previous evening, as well (May 2, 1784), at which the topics on which votes were taken had been discussed. The votes, eleven in total, were all related to the doctors' concerns about John Warren and his involvement with the emerging medical school (now Harvard Medical School), that school's relation to almshouses, the medical care of the poor, and other related matters. The tone and content of these votes reveals anger on the part of the members of the Boston Medical Society towards Warren. This anger appears to have stemmed from the perceived threat of Warren to their own practices, exacerbated by a vote of the Harvard Corporation on April 19, 1784. This vote authorized Warren to apply to the Overseers of the Poor for the town of Boston, requesting that students in the newly-established Harvard medical program, where Warren was Professor of Anatomy and Surgery, be allowed to visit the hospital of the almshouse with their professors for the purpose of clinical instruction. Although Warren believed that the students would learn far more from these visits, in regards to surgical experience, than they could possibly learn in Cambridge, the proposal provoked great distrust from the members of the Boston Medical Society, who accused Warren of an "attempt to direct the public medical business from its usual channels" for his own financial and professional gain.

An essay on the universal plentiude of being and on the nature and immortality of the human soul and its agency : manuscript, [1784]

Autograph manuscript, signed, with autograph manuscript revisions.