We report results of experiments in which we demonstrated the existence of a polymerase I promoter within the ribosomal DNA spacer upstream from the rRNA initiation site in Chinese hamsters and mice. Transcription of the CHO spacer promoter was achieved by the same protein factors, C and D, that catalyzed transcription of the gene promoter, and these factors bound stably to the CHO spacer promoter in a preinitiation complex, just as they did to the gene promoter. In contrast to the CHO spacer promoter, which was transcribed in vitro nearly as efficiently as the gene promoter, the mouse spacer promoter was far less active; this low activity was attributable to the fact that the mouse spacer promoter bound factor D inefficiently. It is striking that the active CHO spacer promoter violated the otherwise universal rule that metazoan RNA polymerase I promoters all have a G residue at position -16. Sequence comparisons also revealed a great similarity between the CHO and mouse spacer promoter regions, yet there was much less similarity between the flanking sequences. There was also only limited homology between the spacer and gene promoter regions, but despite this the two kinds of initiation regions were organized similarly, both consisting of an essential core promoter domain and a stimulatory domain that extended upstream to approximately residue -135. Evolutionary considerations argue strongly that the presence of ribosomal DNA spacer promoters offers a significant selective advantage.Although it is frequently stated that the rRNA gene promoter is the only site at which RNA polymerase I initiates transcription, it has been known for several years that the spacer regions separating adjacent rRNA genes in a number of invertebrate and lower vertebrate species contain repeated elements that are related to the gene promoter. In members of the genera Xenopus, Drosophila, and Artemia, the ribosomal DNA (rDNA) spacer contains two to five spacer promoters whose sequences are highly homologous to those of the respective gene promoters and which exhibit promoter activity in vitro, in vivo, or both (1, 12, 18, 19, 27, 29, 32-34, 37, 43). In Xenopus laevis, both the spacer promoters and adjoining 60-and 81-base-pair (bp) repetitive elements, which are duplications of the central region of the gene promoter, have been shown to affect transcription (6,7,36) and recently have been demonstrated to enhance expression from adjoining rRNA gene promoters (L. Pape, J. Windle, and B. Sollner-Webb, submitted for publication). In addition, an intergenic sequence element in yeast rDNA which has been reported to possess promoter activity in vitro (40) also acts as a yeast rRNA gene enhancer element in vivo (9, 10).In part because of its much greater size, the spacer region of mammalian rDNA repeats remains largely uncharacterized. However, the presence of spacer promoters in many diverse lower organisms suggested that such elements may also exist in mammalian spacer regions. While mouse rDNA evidently does not contain spacer promoters with...

A protein of molecular weight 31,000 became labeled with 32P within 5 min after addition of insulin to differentiated 3T3-L1 preadipocytes previously incubated for 55 min with 32P;. The effect was mimicked by antisera directed against the insulin receptor and was eliminated by anti-insulin antiserum. Incorporation of 32P into this protein was more than 20-fold greater in insulin-treated cells than in cells not exposed to the hormone. At concentrations greater than required with insulin, epidermal growth factor and serum (1-5%) also stimulated phosphorylation whereas I-isoproterenol, a ,i-adrenergic agonist that increases intracellular accumulation of cyclic AMP, was without effect. The 31,000-dalton protein has been tentatively identified as ribosomal protein S6 by two-dimensional polyacrylamide gel electrophoresis. Incorporation of 32P into S6 could be detected within the same time period (5 min) and at the same insulin concentrations (0.1-1.0 nM) as are required to stimulate hexose uptake in both 3T3-L1 cells and mature mammalian adipocytes. The mechanism by which this phosphorylation either mediates or reflects the intracellular actions of insulin remains to be elucidated.During differentiation, the murine preadipocyte cell line 3T3-L1 acquires many of the morphological and biochemical properties of mature mammalian adipocytes (1-6) including a high concentration of cell surface insulin receptors (7,8) and an insulin-sensitive hexose transport system (9, 10). Because the acute effects of insulin on carbohydrate and lipid metabolism in liver, muscle, and adipose tissue impinge, directly or indirectly, on enzyme systems known to be regulated by protein phosphorylation (11,12), the cultured 3T3-L1 cells provide a unique system for (i) investigating insulin-mediated protein phosphorylation in intact cells and (ii) correlating covalent modification of proteins with the physiological effects of insulin. MATERIALS AND METHODSPorcine insulin was a gift from M. Root (Eli Lilly). Carrier-free 32P1 (H332P04) (285 Ci/mg; 1 Ci = 3.7 X 1010 becquerels) was obtained from New England Nuclear, RNase and papain were from Worthington, bovine serum albumin was from Armour Pharmaceutical (Kankakee, IL), cytochalasin B and 1-methyl-3-isobutylxanthine were from Aldrich, acrylamide and bisacrylamide were from Eastman, and ultrapure urea and sucrose were from Schwarz/Mann. Epidermal growth factor was purchased from Collaborative Research (Waltham, MA); anti-porcine insulin serum was a gift from Peter Wright (University of Indiana School of Medicine); rabbit anti-insulin receptor serum was a gift from S. Jacobs and P. Cuatrecasas (Burroughs Wellcome, Research Triangle Park, NC). X-Omat film was purchased from General Electric Co. All other chemicals were from Sigma.Cell For experiments using single-dimension analysis, 5-10% of the processed sample (approximately 50 Mtg of protein) was applied to the gel. The total extract derived from one monolayer was used in the two-dimensional analyses. Sample Preparation. At the conclusion...

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During sporulation of Saccharomyces cerevisiae, most strains accumulate an unmethylated 20S RNA. Contrary to previous reports, this sporulation 20S RNA is distinct from the short-lived methylated 20S RNA precursor of 18S rRNA. This RNA species was found in a cytoplasmic 32S ribonucleoprotein particle consisting of one single-stranded 20S RNA molecule and 18 to 20 identical protein subunits of molecular weight 23,000. The ribonucleoprotein particle was resistant to ribonuclease digestion, although purified 20S RNA was ribonuclease sensitive. Both the RNA and the protein of the 32S ribonucleoprotein particle were only synthesized under conditions that induce sporulation. The accumulation of 20S RNA depended on continued protein synthesis but was actinomycin D insensitive, despite a high guanine-plus-cytosine content. Synthesis of 20S RNA stopped when cells were removed from sporulation conditions and placed in growth medium.M NaCl, and 0.003 M ethylenediaminetetraacetic acid and placed in a chilled Bronwill homogenizing bottle. Glass beads were added in volume equal to the cell 246 on July 31, 2020 by guest

Rhodospirillum rubrum is a facultatively phototrophic bacterium that, under certain growth conditions, forms an intracytoplasmic chromatophore membrane (ICM) housing the photochemical apparatus. The puf operon of R. rubrum encodes protein subunits of the photochemical reaction center and the B880 lightharvesting antenna complex. Mutant strains of R. rubrum were constructed by interposon mutagenesis through which a kanamycin resistance gene cartridge was inserted into restriction sites and in place of restriction fragments of the pufregion. Southern blot analysis demonstrated that the defective copies ofpuf sequences had replaced their normal chromosomal counterparts through homologous recombination. The pheriotypes of the mutant strains were evaluated on the basis of puf gene expression, spectral analysis, pigment content of membranes, and electron-microscopic examination of thin sections of cells grown under semiaerobic and dark anaerobic conditions. Alterations of the puf region affect phototrophic competence and the formation of the ICM. The latter result implies an obligatory role for puf gene products in ICM formation in R. rubrum. One mutant with a deletion in puf structural genes was complemented in trans to the wild-type phenotype. Other mutants could be restored to the wild-type phenotype only by recombination.Rhodospirillum rubrum is a gram-negative, facultatively phototrophic, purple nonsulfur bacterium that grows chemoheterotrophically under aerobic conditions and phototrophically under reduced-oxygen conditions in the light. During the transition from aerobic to reduced-oxygen conditions, photopigments and associated proteins are synthesized and inserted into a differentiated intracytoplasmic chromatophore membrane (ICM). Previous studies have demonstrated that the photosynthetic ICM is physically continuous with (31) and formed by invagination of (16,17,27) the cytoplasmic membrane.Contained within the ICM of R. rubrum are two proteinpigment complexes, the P870 reaction center complex (RC) and the B880 light-harvesting I complex. The RC of R. rubrum consists of three polypeptide subunits, L, M, and H, which are present in a 1:1:1 ratio. Associated with these polypeptides are bacteriochlorophyll (BCHL), bacteriopheophytin, the carotenoid spirilloxanthin (CRT), iron, and ubiquinone (58, 59). R. rubrum has only one antenna complex, the B880 antenna complex, composed of ot and ,B polypeptides, BCHL, CRT, diphosphatidylethanolamine, and diphosphatidylglycerol (50).The genes encoding the B880 ot and polypeptides, and those encoding the reaction center L and M polypeptides, have been cloned and sequenced in Rhodobacter capsulatus (66, 67), Rhodobacter sphaeroides (35, 61, 62), R. rubrum (7, 10), and Rhodopseudomonas viridis (40, 60). These genes are closely linked in a single operon termed puf. The pufL and pufM genes of R. rubrum, Rhodopseudomonas viridis, Rhodobacter sphaeroides, and Rhodobacter capsulatus show a nucleotide sequence similarity ranging from 61 to 78% (7,10,35,60,66,67). R. rubrum puf seq...