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  • 1
    ISSN: 0170-2041
    Keywords: Housane ; Norcaradiene ; Chemistry ; Organic Chemistry
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Cycloaddition of a Very Reactive Cyanovinylcarbene with Benzene and 3,4-Dichlorocyclobutene. Molecular and Crystal Structure of 2,3-Dichloro-5-(1-cyano-2-methyl-1-propenyl)-5-housanecarbonitrile and 7-(1-Cyano-2-methyl-1-propenyl)-7-norcaradienecarbonitrileThe cyanovinylcarbene 2 has been generated by photolysis of 3,3-dimethyl-3H-pyrazole-4,5-dicarbonitril (1) and the cycloaddition products with benzene and with 3,4-dichlorocyclobutene have been isolated. The molecular structures of the cycloaddition products 7-(1-cyano-2-methyl-1-propenyl)-7-norcaradienecarbonitrile (3) and 2,3-dichloro-5-(1-cyano-2-methyl-1-propenyl)-5-housanecarbonitrile (4) were determined by X-ray analyses. The bridging bond of the bicyclo[2.1.0]pentane group in 4 is shortened to 1.515 Å by the electronic interaction of this group with the cyano substituent. The vinyl substituent has no influence because of perpendicular orientation.
    Additional Material: 2 Ill.
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  • 2
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; HEM13 regulation ; Heme and oxygen ; CYP1, ROX1, SSN6, TUP1
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Saccharomyces cerevisiae HEM13 gene codes for coproporphyrinogen oxidase (CPO), an oxygen-requiring enzyme catalysing the sixth step of heme biosynthesis. Its transcription is increased 40–50-fold in response to oxygen- or heme-deficiency. We have analyzed CPO activity and HEM13 mRNA levels in a set of isogenic strains carrying single or double deletions of the CYP1 (HAP1), ROX1, SSN6, or TUPI genes. The cells were grown in the presence or absence of oxygen and under heme-deficiency (hem1Δ background). Both Rox1p and Cyp1p partially repressed HEM13 in aerobic heme-sufficient cells, probably in an independent manner. In the absence of heme, Cyp1p activated HEM13 and strongly repressed ROX1, allowing de-repression of HEM13. Cyp1p had no effect on HEM13 expression in anaerobic cells. Deletions of SSN6 or TUP1 dramatically de-repressed HEM13 in aerobic cells. A series of deletions in the HEM13 promoter identified at least four regulatory regions that are required for HEM13 regulation. Two regions, containing motifs similar to the Rox1p consensus sequences, act as repression sites under aerobic growth. The two other sites act as activation sequences required for full induction under oxygen- or heme-deficiency. Taken together, these results suggest that induction of HEM13 occurs in part through relief of repression exerted by Rox1p and Cyp1p, and in part by activation mediated partly by Cyp1p under heme-deficiency and by unknown factors under oxygen-deficiency.
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  • 3
    ISSN: 1432-0983
    Keywords: Yeast ; Mitochondria ; Aminoacyl-tRNA synthetase ; RNA splicing
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The mitochondrial leucyl-tRNA synthetase (mLRS) of Saccharomyces cerevisiae is involved in both mitochondrial protein synthesis and pre-mRNA splicing. We have created mutations in the regions HIGH, GWD and KMSKS, which are involved in ATP-, amino acid-and tRNA-binding respectively, and which have been conserved in the evolution of group I tRNA synthetases. The mutants GRD and NMSKS have no discernible phenotype. The mutants AWD and ARD act as null alleles and lead to the production of 100% cytoplasmic petites. The mutants HIGN, NIGH and KMSNS are unable to grown on glycerol even in the presence of an intronless mitochondrial genome and accumulate petites to a greater extent than the wild-type but less than 40%. Experiments with an imported bI4 maturase indicate that the lesion in these mutations primarily affects the synthetase and not the splicing functions.
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  • 4
    ISSN: 1617-4623
    Keywords: Aminoacyl-tRNA synthetase ; RNA splicing ; Group I introns ; RNA maturase ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The Saccharomyces cerevisiae nuclear gene NAM2 codes for mitochondrial leucyl-tRNA synthetase (mLRS). Herbert et al. (1988, EMBO J 7:473–483) proposed that this protein is involved in mitochondrial RNA splicing. Here we present the construction and analyses of nine mutations obtained by creating two-codon insertions within the NAM2 gene. Three of these prevent respiration while maintaining the mitochondrial genome. These three mutants: (1) display in vitro a mLRS activity ranging from 0%–50% that of the wild type: (2) allow in vivo the synthesis of several mitochondrially encoded proteins; (3) prevent the synthesis of the COXII protein but not of its mRNA; (4) abolish the splicing of the group I introns bI4 and aI4; and (5) affect significantly the excision of the group I introns bI2, bI3 and aI3. Importation of the bI4 maturase from the cytoplasm into mitochondria in a nam2 − mutant strain does not restore the excision of the introns bI4 and aI4 implying that the splicing deficiency does not result from the absence of the bI4 maturase. We conclude that the mLRS is a splicing factor essential for the excision of the group I introns bI4 and aI4 and probably important for the excision of other group I introns.
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  • 5
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Cell cycle ; Bud site selection ; Guanine exchange factor ; Ras
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Guanine Exchange Factor (GEF) activity for Ras proteins has been associated with a conserved domain in Cdc25p, Sdc25p in Saccharomyces cerevisiae and several other proteins recently found in other eukaryotes. We have assessed the structure-function relationships between three different members of this family in S. cerevisiae, Cdc25p, Sdc25p and Bud5p. Cdc25p controls the Ras pathway, whereas Bud5p controls bud site localization. We demonstrate that the GEF domain of Sdc25p is closely related to that of Cdc25p. We first constructed a thermosensitive allele of SDC25 by specifically altering amino acid positions known to be changed in the cdc25-1 mutation. Secondly, we constructed three chimeric genes from CDC25 and SDC25, the products of which are as active in the Ras pathway as are the wild-type proteins. In contrast, similar chimeras made between CDC25 and BUD5 lead to proteins that are inactive both in the Ras and budding control pathways. This difference in the ability of chimeric proteins to retain activity allows us to define two subclasses of structurally different GEFs: Cdc25p and Sdc25p are Ras-specific GEFs, and Bud5p is a putative GEF for the Rsr1/Bud1 Rap-like protein.
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  • 6
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Actin cytoskeleton ; Budding pattern ; Amphiphysin ; Myosin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The actin cytoskeleton cells is altered in rvs161 mutant yeast, with the defect becoming more pronounced under unfavorable growth conditions, as described for the rvs167 mutant. The cytoskeletal alteration has no apparent effect on invertase secretion and polarized growth. Mutations in RTVS161, just as in RI/S167, lead to a random budding pattern in a/α diploid cells. This behavior is not observed in a/a diploid cells homozygous for the rvs161-1 or rvs167-1 mutations. In addition, sequence comparisons revealed that amphiphysin, a protein first found in synaptic vesicles of chicken and shown to be the autoantigen of Stiff Man syndrome, presents similarity with both Rvs proteins. Furthermore, limited similarities with myosin heavy chain and tropomyosin alpha chain from higher eukaryotic cells allow for the definition of a possible consensus sequence. The finding of related sequences suggests the existence of a function for these proteins that is conserved among eukaryotic organisms.
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  • 7
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Translation ; Splicing ; Paromomycin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The MSS51 gene product has been previously shown to be involved in the splicing of the mitochondrial pre-mRNA of cytochrome oxidase subunit I (COX1). We show here that it is specifically required for the translation of the COX1 mRNA. Furthermore, the paromomycin-resistance mutation (P inf454 supR ) which affects the 15 S mitoribosomal RNA, interferes, directly or indirectly, with the action of the MSS51 gene product. Possible roles of the MSS51 protein on the excision of COX1 introns are discussed.
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  • 8
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Cell cycle ; Proline ; DNA sequencing
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We report here the isolation of temperature-sensitive mutants of the yeast Saccharomyces cerevisiae which exhibit cdc phenotypes. The recessive mutations defined four complementation groups, named ore1, ore2, ore3 and ore4. At the non-permissive temperature, strains bearing these mutations arrested in the G1 phase of the cell cycle. The wild-type allele of the gene altered in ore2 mutants was cloned. The nucleotide sequence of a fragment which can complement the mutation showed the presence of an open reading frame capable of encoding a protein with 286 amino acid residues. The deduced amino acid sequence showed 25% identity with that of the Escherichia coli Δ1-pyrroline-5-carboxylate reductase, an enzyme of the pathway for the biosynthesis of proline. The ore2 mutants, correspondingly, were found to be capable of growing at the non-permissive temperature on a synthetic medium supplemented with proline. In addition, the chromosomal location of the gene and its restriction map were compatible with those previously reported for the PRO3 gene which encodes the S. cerevisiae Δ1-pyrroline-5-carboxylate reductase.
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  • 9
    ISSN: 0899-0042
    Keywords: simulated moving bed technology ; chiral separation ; cellulose triacetate ; preparative scale liquid chromatography ; racemic epoxide ; Chemistry ; Organic Chemistry
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The feasibility of using simulated moving bed technology (SMB) for chiral separation on cellulose triacetate is demonstrated on the preparative scale: 1 kg of a chiral epoxide has been separated. On comparing SMB technology with conventional liquid chromatography it turns out that the main advantage of SMB lies in the significant reduction of mobile phase consumption. The process design for SMB is made theoretically and the predictions are confirmed by our pilot study. © 1993 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
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  • 10
    ISSN: 0899-0042
    Keywords: propranolol ; enantiomers ; immunogen synthesis ; selective antibody ; ELISA ; Chemistry ; Organic Chemistry
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: A selective antibody to (S)-propranolol enantiomer was produced in rabbits by immunization with a new conjugate of N-aminopropylpropranolol-albumin. A hapten was first prepared by condensing (S)-propranolol or the racemate with 3-bromopropylphthalimide followed by hydrazinolysis, and the resulting compound conjugated to serum albumin by means of a glutaraldehyde- or carbodiimide-mediated reaction. Rabbits were immunized, and titres and specificity of antibodies were determined by ELISA. The antibodies obtained were tested with (S)-, (R)-, (R, S)-propranolol, and other structural analogs. Selective (S)-antibodies recognized (S)-propranolol 20 times more avidly than (R)-isomer while an antiserum against (R, S)-propranolol recognized both (S)- and (R)-isomers to about the same degree. ©1993 Wiley-Liss, Inc.
    Additional Material: 3 Ill.
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