Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae.

In this paper we show that a 211-base pair segment of CEN3 DNA is sufficient to confer wild-type centromere function in the yeast Saccharomyces cerevisiae. We used site-directed mutagenesis of the 211-base pair fragment to examine the sequence-specific functional requirements of a conserved 11-base pair segment of centromere DNA, element III (5'-TGATTTATCCGAA-3'). Element III is the most highly conserved of the centromeric DNA sequences, differing by only a single adenine X thymine base pair among the four centromere DNAs sequenced thus far. All of the element III sequences contain specific cytosine X guanine base pairs, including a 5'-CCG-3' arrangement, which we targeted for single cytosine-to-thymine mutations by using sodium bisulfite. The effects of element III mutations on plasmid and chromosome segregation were determined by mitotic stability assays. Conversion of CCG to CTG completely abolished centromere function both in plasmids and in chromosome III, whereas conversion of CCG to TCG decreased plasmid and chromosome stability moderately. The other two guanine X cytosine base pairs in element III could be independently converted to adenine X thymine base pairs without affecting plasmid or chromosome stability. We concluded that while some specific nucleotides within the conserved element III sequence are essential for proper centromere function, other conserved nucleotides can be changed.     

Human alveolar macrophage fibronectin: synthesis, secretion, and ultrastructural localization during gelatin-coated latex particle binding

Human pulmonary alveolar macrophages synthesized and secreted several characteristic high molecular weight proteins for at least 7 d in vitro. Immunoprecipitates of medium and cell lysates from metabolically labeled cultures with specific anti-human plasma fibronectin IgG contained one major labeled polypeptide of molecular weight 440,000 (unreduced) or 220,000 (reduced). An identical polypeptide in conditioned medium from radiolabeled macrophages bound specifically to gelatin-Sepharose, demonstrating that alveolar macrophages synthesized and secreted a molecule immunologically and functionally similar to fibronectin. Fibronectin was the major newly synthesized and secreted polypeptide of freshly harvested alveolar macrophages. Pulse-chase experiments revealed that newly synthesized fibronectin was rapidly secreted into medium, approximately 50 percent appearing by 1 h and 80 percent by 8 h. Immunoperoxidase staining using antifibronectin F(ab’)(2)-peroxidase conjugates revealed the majority of immunoreactive fibronectin to be intracellular, localized to endoplasmic reticulum and Golgi apparatus. No extracellular matrix fibronectin was visualized, and cell surface staining was rarely seen, usually appearing only at sites where cells were closely apposed and not at sites of macrophage-substrate attachment. Similar immunostaining of fibroblast cultures revealed cell surface-associated fibrillar fibronectin. Ultrastructural localization of fibronectin during binding and phagocytosis of gelatin-coated and plain latex particles revealed fibronectin only on gelatin-latex beads and at their cell binding sites. Neigher plain latex beads nor their cell membrane binding sites stained for fibronectin. These results demonstrate that fibronectin is a major product of human alveolar macrophages, is rapidly secreted, and is localized at cell membrane binding sites for gelatin-coated particles. In view of the known binding properties of fibronectin, it may serve as an endogenous opsonic factor promoting the binding of staphylococcus, denatured collagen, fibrin, or other macromolecules to macrophages in the lower respiratory tract.     

Cellular differentiation and morphogenesis in Cordylophora

Summary The interstitial cells ofCordylophora were destroyed by treating animals with 4,500 Roentgens of x-irradiation. Within 5–6 days after treatment no interstitial cells were detected in the treated animals and they were never seen in later stages. Some cell divisions were noted in the epidermal epithelio-muscular cells of the x-rayed animals which survived for four weeks. This was ample time to perform reaggregation-reconstitution experiments.Isolated, untreated coenosarc formed a mass from which hydranths and stolons arose. X-rayed coenosarc also formed these structures, although regenerative capacity was less than that of normal coenosarc. The number of stolons and hydranths produced decreased with length of time after irradiation. Both normal and x-rayed coenosarc masses exhibited a tendency to form a greater number of hydranths than stolons when the ratio of epidermis to gastrodermis was low and a greater number of stolons when the ratio of epidermis to gastrodermis was high. Masses prepared from the amounts of epidermis and gastrodermis normally found in intact animals produced intermediate numbers of hydranths and stolons.Isolated, untreated epidermis produced a gastrodermal layer from interstitial cells. They migrated to the inner surface of the epidermal epithelio-muscular cells, enlarged and differentiated into typical gastrodermal-digestive cells. These preparations formed hydranths and developed into colonies. X-irradiated epidermis did not form an inner gastrodermal layer but did secrete perisarc on the periphery. In some ases a second layer of epidermal epithelio-muscular cells was noted on the interior of the x-rayed masses. However, none of the irradiated epidermal masses produced hydranths or stolons or survived to form colonies.Gastrodermis was isolated from normal animals and although the cells rounded up into a spherical mass no morphogenesis occurred and the masses disintegrated within 12–24 hours. Irradiated gastrodermis behaved in the same manner.Normal epidermis was applied to x-rayed gastrodermis and from these preparations normal animals were produced.Normal, untreated gastrodermis combined with x-rayed epidermis yielded viable animals. Interstitial cells appeared to be produced by dedifferentiation of gland cells. The interstitial cells thus formed were able to divide and differentiate into cnidoblasts typical of epidermis.Thus, inCordylophora both epidermal and gastrodermal cells have the capacity to form cell types characteristic of the reciprocal layer.

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Zusammenfassung Die Interstitialzellen vonCordylophora wurden durch Röntgenbestrahlung (4,500 r) zerstört. 5–6 Tage nach der Bestrahlung waren keine Interstitialzellen mehr feststellbar, und auch in späteren Stadien traten nie mehr solche auf. Einige Zellteilungen wurden in den Epithel-Muskelzellen der bestrahlten Tiere beobachtet. Die Tiere blieben 4 Wochen am Leben, so daß Reaggregations- und Rekonstitutionsexperimente durchgeführt werden konnten.Isoliertes, unbehandeltes Coenosark bildete eine Zellmasse, aus der heraus Hydranthen und Stolonen wuchsen. Dieselben Strukturen entstanden aus bestrahltem Coenosark, wenn auch die Regenerationsfähigkeit des bestrahlten Cornosarks vermindert war. Die Zahl der gebildeten Hydranthen und Stolonen nahm mit zunehmender Zeit nach der Bestrahlung ab. Normale und auch bestrahlte Coenosark-Massen bildeten im allgemeinen mehr Hydranthen als Stolonen, wenn das Verhältnis Epidermis: Gastrodermis niedrig war, aber mehr Stolonen, wenn dag Verhältnis hoch war. Coenosark-Massen, die Epidermis und Gastrodermis im normalerweise vorhandenen Verhältnis enthielten, bildeten intermediäre Zahlen von Hydranten und Stolonen aus.Isolierte, unbehandelte Epidermis bildete aus Interstitialzellen eine Gastrodermisschicht. Dabei wanderten die Interstitialzellen auf die innere Oberfläche der epidermalen Epithelmuskelzellen, wurden größer, und bildeten typische gastrodermale Verdauungszellen. Solche Präparate bildeten Hydranthen und entwickelten sich in Kolonien. Bestrahlte Epidermis bildete keine Gastrodermis aus, sezernierte aber Perisark auf der Peripherie. In einigen Fällen wurde eine zweite Schicht von epidermalen Epithelmuskelzellen beobachtet im Innern der bestrahlten Massen. Me aber bildete eine bestrahlte Epidermismasse Hydranthen oder Stolonen, und nie Kolonien.Wenn Gastrodermis aus normalen Tieren isoliert wurde, rundeten sich die Zellen sich zu einer kugeligen Masse ab, aber es kam nie zu einer Morphogenese, und die Massen desintegrierten innerhalb 12–24 Std. Bestrahlte Gastrodermis verhielt sich ebenso.Wenn normale Epidermis auf bestrahlte Gastrodermis gepflanzt wurde, entstanden normale Tiere.Wenn normale Gastrodermis mit bestrahlter Epidermis kombiniert wurde, entstanden ebenfalls normale Tiere. Interstitialzellen entstanden durch Dedifferenzierung von Drüsenzellen. Die dabei gebildeten Interstitialzellen teilten sich und bildeten für Epidermis typische Cnidoblasten. Bei Cordylophora haben demzufolge epidermale, als auch gastrodermale Zellen die Fähigkeit, Zellen der reziproken Zellschicht auszubilden.

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The author wishes to acknowledge the financial aid received for this project from the National Institutes of Health, Bethesda, Md. through their University Biomedical Sciences Support Program.     

Dicyclohexylammonium salts for the isolation and characterization of aldonic acids

Dicyclohexylammonium salts of aldonic acids may be prepared from aldonolactones, as well as from metal aldonates and the free acids. Although accompanied by decomposition, their melting points are usually sharp, and these salts appear to have some potential utility for the isolation and characterization of aldonic acids.Dicyclohexylammonium 2-acetamido-2-deoxy-d-gluconate (1) has recently been described; in the course of the present investigation, it was converted into 2-acetamido-2-deoxy-d-glucose (3), confirming the configuration previously assigned to it. With aqueous dicyclohexylamine, 2-acetamido-2-deoxy-d-mannono-1,4-lactone (2) gives 1. The configuration of 2 was reconfirmed through reduction to 2-acetamido-2-deoxy-d-mannitol (4), and the optical rotations of this compound and its d-gluco isomer in acidified ammonium molybdate solution were found to be useful physical constants for distinguishing these alditols.2-Acetamido-2-deoxy-d-galactono-1,4-lactone affords a crystalline dicyclohexylammonium salt of the corresponding acid, from which the lactone may be regenerated.     

Structural homology among mammalian and Saccharomyces cerevisiae isoprenyl-protein transferases

Farnesyl-protein transferase (FTase) purified from rat or bovine brain is an alpha/beta heterodimer, comprised of subunits having relative molecular masses of approximately 47 (alpha) and 45 kDa (beta). In the yeast Saccharomyces cerevisiae, two unlinked genes, RAM1/DPR1 (RAM1) and RAM2, are required for FTase activity. To explore the relationship between the mammalian and yeast enzymes, we initiated cloning and immunological analyses. cDNA clones encoding the 329-amino acid COOH-terminal domain of bovine FTase alpha-subunit were isolated. Comparison of the amino acid sequences deduced from the alpha-subunit cDNA and the RAM2 gene revealed 30% identity and 58% similarity, suggesting that the RAM2 gene product encodes a subunit for the yeast FTase analogous to the bovine FTase alpha-subunit. Antisera raised against the RAM1 gene product reacted specifically with the beta-subunit of bovine FTase, suggesting that the RAM1 gene product is analogous to the bovine FTase beta-subunit. Whereas a ram1 mutation specifically inhibits FTase, mutations in the CDC43 and BET2 genes, both of which are homologous to RAM1, specifically inhibit geranylgeranyl-protein transferase (GGTase) type I and GGTase-II, respectively. In contrast, a ram2 mutation impairs both FTase and GGTase-I, but has little effect on GGTase-II. Antisera that specifically recognized the bovine FTase alpha-subunit precipitated both bovine FTase and GGTase-I activity, but not GGTase-II activity. Together, these results indicate that for both yeast and mammalian cells, FTase, GGTase-I, and GGTase-II are comprised of different but homologous beta-subunits and that the alpha-subunits of FTase and GGTase-I share common features not shared by GGTase-II.