Biosci Biotechnol Biochem, 1995 Jul, 59(7), 1345 - 8 Nucleotide sequence of alpha-galactosidase cDNA from Mortierella vinacea; Shibuya H et al.; To analyze the primary structure of Mortierella vinacea alpha-galactosidase, a cDNA library of M . vinacea mRNA in lambda gt10 was constructed . A clone, which has an insert size of about 1.4 kilobase pairs, was found to contain the coding region of the mature enzyme . The deduced amino acid sequence share that the mature enzyme consisted of 397 amino acid residues with a molecular mass of 44,350 Da . The sequence identity of the mature enzyme with alpha-galactosidases from Saccharomyces carlsbergensis, Cyamopsis tetragonoloba (guar), and human were 47%, 43%, and 34%, respectively. Bioessays, 1995 Jun, 17(6), 553 - 6 Drosophila development pulls the strings of the cell cycle; Reed BH; The three cycles of cell division immediately following the formation of the cellular blastoderm during Drosophila embryogenesis display an invariant pattern . Bursts of transcription of a gene called string are required and sufficient to trigger mitosis at this time during development . The activator of mitosis encoded by the string gene is a positive regulator of cdc2 kinase and a Drosophila homologue of the Saccharomyces pombe cdc25 tyrosine phosphatase . Evidence presented in a recent paper demonstrates that transcription of string, and hence the timing and pattern of mitosis in the postblastoderm embryo, is under complex developmental control . Several lines of evidence support this interpretation, including the analysis of string transcription in pattern formation mutants, cell cycle arrest mutants, and the preliminary characterization of an extensive cis-acting regulatory region. Mol Biol Evol, 1995 May, 12(3), 405 - 14 The novel flightless-I gene brings together two gene families, actin-binding proteins related to gelsolin and leucine-rich-repeat proteins involved in Ras signal transduction; Claudianos C et al.; The Drosophila melanogaster gene flightless-I, involved in gastrulation and muscle degeneration, has Caenorhabditis elegans and human homologues . In these highly conserved genes, two previously known gene families have been brought together, families encoding the actin-binding proteins related to gelsolin and the leucine-rich-repeat (LRR) group of proteins involved in protein-protein interactions . Both these gene families exhibit characteristics of molecular changes involving replication slippage and exon shuffling . Phylogenetic analyses of 19 amino acid sequences of 6 related protein types indicate that actin-associated proteins related to gelsolin are monophyletic to a common ancestor and include flightless proteins . Conversely, comparison of 24 amino acid sequences of LRR proteins including the flightless proteins indicates that flightless proteins are members of a structurally related subgroup . Included in the flightless cluster are human and mouse rsp-1 proteins involved in suppressing v-Ras transformation of cells and the membrane-associated yeast (Saccharomyces cerevisae) adenylate cyclase whose analogous LRRs are required for interaction with Ras proteins . There is a strong possibility that ligands for this group could be related and that flightless may have a similar role in Ras signal transduction . It is hypothesized that an ancestral monomeric gelsolin precursor protein has undergone at least four independent gene reorganization events to account for the structural diversity of the extant family of gelsolin-related proteins and that gene duplication and exon shuffling events occurred prior to or at the beginning of multicellular life, resulting in the evolution of some members of the family soon after the appearance of actin-type proteins. J Biol Chem, 1995 Apr 28, 270(17), 10351 - 7 Identification by targeted differential display of an immediate early gene encoding a putative serine/threonine kinase; Donohue PJ et al.; Fibroblast growth factor (FGF)-1 mitogenic signal transduction is mediated in part by gene products that are specifically expressed in response to cell surface receptor binding and activation . We have used a targeted differential display method to identify FGF-1-inducible genes in murine NIH 3T3 fibroblasts . Here we report that one of these genes is predicted to encode a novel serine/threonine-specific protein kinase . This putative kinase has been named Fnk, for FGF-inducible kinase . The deduced Fnk amino acid sequence has 49, 36, 33, 32, and 22% overall identity to mouse serum-inducible kinase (Snk), mouse polo-like kinase (Plk), Drosophila polo, Saccharomyces Cdc5, and mouse Snk/Plk-akin kinase (Sak), respectively . These proteins are all members of the polo subfamily of structurally related serine/threonine kinases . The Plk, polo, Cdc5, and Sak kinases are required for cell division . FGF-1 induction of Fnk mRNA expression is first detected at 30 min after mitogen addition, reflects transcriptional activation, and does not require de novo protein synthesis . FGF-2, platelet-derived growth factor-BB, calf serum, or phorbol myristate acetate treatment of quiescent cells also induces fnk gene expression . Fnk mRNA is expressed in vivo in a tissue-specific manner, with relatively high levels detected in newborn and adult mouse skin . These results indicate that Fnk may be a transiently expressed protein kinase involved in the early signaling events required for growth factor-stimulated cell cycle progression. Yeast, 1995 Apr 15, 11(4), 327 - 36 Glucose metabolism, enzymic analysis and product formation in chemostat culture of Hanseniaspora uvarum; Venturin C et al.; The physiology of Hanseniaspora uvarum K5 was studied in glucose-limited chemostat cultures and upon glucose pulse . Up to a dilution rate of 0.28 h-1, glucose was completely metabolized in biomass and CO2 . Above this value, increase in the dilution rate was accompanied by sequential production of metabolites (glycerol, acetate and ethanol) and decrease in cell yield . Similar results were observed upon glucose pulse . From the enzyme activities (pyruvate dehydrogenase, pyruvate decarboxylase, NAD and NADP-dependent acetaldehyde dehydrogenases, acetyl coenzyme A synthetase and alcohol dehydrogenase) and substrate affinities, the following conclusions were drawn with respect to product formation of cells: (1) pyruvate was preferentially metabolized via pyruvate dehydrogenase, when biomass and CO2 were the only products formed; (2) acetaldehyde formed by pyruvate decarboxylase was preferentially oxidized in acetate by NADP-dependent aldehyde dehydrogenase; acetate accumulation results from insufficient activity of acetyl-CoA synthetase required for the complete oxidation of acetate; (3) acetaldehyde was oxidized in ethanol by alcohol dehydrogenase, in addition to acetate production. J Biochem (Tokyo), 1995 Apr, 117(4), 863 - 8 Analysis of a conformation-independent epitope and a conformational epitope in a protein: a study on cobrotoxin from Taiwan cobra venom; Chang LS et al.; The antibodies against cobrotoxin were separated into two antibody preparations by successive affinity chromatographies on reduced and S-carboxymethylated (RCM)-cobrotoxin-Sepharose, and cobrotoxin-Sepharose columns . The antibodies (abbreviated as Abcf-i) that bound with the RCM-cobrotoxin-Sepharose were verified to specifically recognize the continuous epitopes of cobrotoxin, which were insensitive to conformational changes . Whilst the antibodies (abbreviated as Abcf-d) that did not bind with the RCM-cobrotoxin-Sepharose column recognized the conformational epitopes in cobrotoxin . The two antibody preparations were employed to screen the antigenic peptides derived from the proteolytic hydrolysate of cobrotoxin and RCM-cobrotoxin . Five antigenic peptides (AP-4, AP-5, AP-10, AP-11, and AP-12) were obtained from the acid protease A-digested hydrolysate of cobrotoxin, and two antigenic peptides (V8-2 and V8-4) were found in the hydrolysate of RCM-cobrotoxin after hydrolysis with Saccharomyces aureus V8 protease . The segments at positions 1-21 and 22-38 encompassed the peptide fractions, AP-4, AP-5, V8-2, and V8-4, that reacted with Abcf-i, indicating that the two segments bore the continuous epitopes of cobrotoxin . Alternatively, AP-10, AP-11, and AP-12 reacted with both Abcf-i and Abcf-d . The structures of the three peptides had a common segment at positions 43-62, suggesting that this region comprised the conformation-independent epitopes as well as conformational epitopes in cobrotoxin . These results reflected that the conformation-independent and conformational epitopes in a protein can be separately identified. Biochim Biophys Acta, 1995 Mar 14, 1261(1), 147 - 50 Molecular cloning of the Drosophila homologue of the rat ribosomal protein L11 gene; Larochelle S et al.; We report the isolation of the Drosophila melanogaster homologue of the rat ribosomal protein L11 gene . The gene is present in the Drosophila genome at polytene chromosome location 56D, on the right arm of the second chromosome . The Drosophila DL11 gene appears to encode two messages of 0.8 and 0.9 kb which are expressed throughout development with variations in their relative abundance . DL11 codes for a predicted protein of 184 amino acids with a molecular mass of 21.1 kDa. Genetics, 1995 Mar, 139(3), 1449 - 54 Estimating interference and linkage map distance from two-factor tetrad data; Stahl FW et al.; We present methods for using the model of Foss, Lande, Stahl and Steinberg to estimate interference and map distances from two-factor tetrad data . We illustrate the application of the methods with data from Neurospora and from Saccharomyces. J Biol Chem, 1995 Jan 20, 270(3), 1429 - 36 The Yarrowia lipolytica gene PAY2 encodes a 42-kDa peroxisomal integral membrane protein essential for matrix protein import and peroxisome enlargement but not for peroxisome membrane proliferation; Eitzen GA et al.; PAY genes are required for peroxisome assembly in the yeast Yarrowia lipolytica . Here we show that a mutant strain, pay2, is disrupted for the import of proteins targeted by either peroxisomal targeting signal-1 or -2 . Electron microscopy of pay2 cells revealed the presence of small peroxisomal "ghosts," similar to the vesicular structures found in fibroblasts of patients with the human peroxisome assembly disorder, Zellweger syndrome . Functional complementation of pay2 with a plasmid library of Y . lipolytica genomic DNA identified a gene, PAY2, that restores growth of pay2 on oleic acid, import of catalase and multifunctional enzyme into peroxisomes, and formation of wild type peroxisomes . The PAY2 gene encodes Pay2p, a hydrophobic polypeptide of 404 amino acids . An antibody raised against Pay2p recognizes a polypeptide of approximately 42-kDa whose synthesis is induced by growth of Y . lipolytica on oleic acid . Pay2p is a peroxisomal integral membrane protein, as it localizes to carbonate-stripped peroxisomal membranes . Pay2p shows no identity to any known protein . Our results suggest that Pay2p is essential for the activity of the peroxisomal import machinery but does not affect the initial steps of peroxisomal membrane proliferation. Antonie Van Leeuwenhoek, 1995, 67(2), 177 - 9 Babjevia gen . nov.--a new genus of the Lipomycetaceae; Smith MT et al.; The species described as Lipomyces anomalus Babjeva & Gorin shows significant genetic and phenotypic divergence from the type species Lipomyces starkeyi Lodder & Kreger-van Rij in terms of rRNA base sequence substitution and ascosporal and septal ultrastructure . The species is consequently reclassified in the new, unispecific genus Babjevia, as Babjevia anomala. Biochimie, 1995, 77(1-2), 22 - 9 Classical and novel approaches to the detection and localization of the numerous modified nucleotides in eukaryotic ribosomal RNA; Maden BE et al.; Human ribosomes contain more than 200 modified nucleotides . These are made up as follows: more than 100 2'-O-methyl groups, 10 methylated bases, about 95 pseudouridines and at least one other modification . Other mammalian sources that have been examined, as well as the lower vertebrate Xenopus laevis, show very similar patterns of nucleotide modifications, especially as revealed by oligonucleotide fingerprinting for methyl groups . Most of the methyl groups have been located along the rRNA primary structure by matching oligonucleotide sequence data to the complete sequences derived from rDNA . Nearly all of the methyls are in conserved core regions . Saccharomyces carlsbergensis ribosomes contain about 55% as many methyls as vertebrate ribosomes . The locations of most of the S carlsbergensis methyls are also known . However, of the numerous other eukaryotes whose rRNA sequences have been determined indirectly from rDNA, few have yielded detailed data on modified nucleotides . This is in part because the methods applied to vertebrate and yeast ribosomes are highly laborious and are not universally applicable . Therefore in the final part of this paper we briefly review other methods that have been applied to the detection and localization of modified nucleotides in rRNA . In particular, we outline progress towards developing a method whereby reverse transcription shows characteristic pausing at most of the 2'-O-methylation sites in human and Xenopus 18S rRNA . 2'-O-Methylation pauses are distinguishable from most other interruptions; the 2'-O-methyl pauses occur more strongly at low than at high dNTP concentration, whereas most other interruptions are independent of dNTP concentration. J Am Soc Nephrol, 1994 Dec, 5(6), 1288 - 99 Activation of Ras by receptor tyrosine kinases; Margolis B et al.; Ras, a small GTP-binding protein, is an important component of the signal transduction pathway used by growth factors to initiate cell growth and differentiation . Cell activation with growth factors such as epidermal growth factor (EGF) induces Ras to move from an inactive GDP-bound state to an active GTP-bound state . Recently, a combination of genetic and biochemical studies has resulted in the elucidation of a signaling pathway that leads from growth factor receptors to Ras . After binding EGF, the EGF receptor tyrosine kinase is activated, leading to receptor autophosphorylation on multiple tyrosine residues . Signaling proteins with Src homology 2 (SH2) domains then bind to these tyrosine-phosphorylated residues, initiating multiple signaling cascades . One of these SH2 domain proteins, Grb2, exists in the cytoplasm in a preformed complex with a second protein, Son of Sevenless (Sos), which can catalyze Ras GTP/GDP exchange . After growth factor stimulation, the tyrosine phosphorylated EGF receptor binds the Grb2/Sos complex, translocating it to the plasma membrane . This translocation is thought to bring Sos into close proximity with Ras, leading to the activation of Ras . In contrast, the insulin receptor does not bind Grb2 directly but rather induces the tyrosine phosphorylation of two proteins, insulin receptor substrate-1 and Shc, that bind the Grb2/Sos complex . Once Ras is activated, it proceeds to stimulate a cascade of protein kinases that are important in a myriad of growth factor responses. Appl Environ Microbiol, 1994 Dec, 60(12), 4324 - 31 PCR primers that allow intergeneric differentiation of ascomycetes and their application to Verticillium spp; Li KN et al.; A pair of conserved PCR primers, designated NMS1 and NMS2, that amplify a region in the mitochondrial small rRNA gene region were designed for fungi belonging to the class Ascomycetes . These primers were tested with members of eight fungal genera (Aspergillus, Fusarium, Magnaporthe, Mycospharella, Neurospora, Saccharomyces, Sclerotinia, Verticillium) and 10 Verticillium species (Verticillium albo-atrum, Verticillium chlamydosporium, Verticillium cinnebarium, Verticillium dahliae, Verticillium fungicola, Verticillium lecanii, Verticillium lateritium, Verticillium nigrescens, Verticillium psaliotae, and Verticillium tricorpus) . The primers were also tested with 35 isolates of V . dahliae obtained from diverse geographic areas and diverse hosts . The results of a restriction fragment length polymorphism analysis of the region amplified by the primers differentiated the genera examined and the results of a DNA sequence analysis of the amplified region differentiated the Verticillium species . Two Fusarium species were also differentiated by the results of the restriction fragment length polymorphism analysis . On the basis of the nucleotide sequences of the amplified regions, we obtained a pair of PCR primers that could be used to differentiate V . dahliae from the other fungal isolates tested, including V . albo-atrum, a closely related plant-pathogenic species . The V . dahliae-specific PCR primer may aid in more rapid and specific detection of the pathogen directly in plant and/or soil samples . PCR primers NMS1 and NMS2 may be used as potential mitochondrial markers for studying fungal cytoplasmic inheritance of ascomycetes and for identifying DNA probes that are informative at or below the genus level. Structure, 1994 Nov 15, 2(11), 1089 - 105 Old yellow enzyme at 2 A resolution: overall structure, ligand binding, and comparison with related flavoproteins; Fox KM et al.; BACKGROUND: Old yellow enzyme (OYE) was the first flavoenzyme purified, but its function is still unknown . Nevertheless, the NADPH oxidase activity, the flavin mononucleotide environment and the ligand-binding properties of OYE have been extensively studied by biochemical and spectroscopic approaches . Full interpretation of these data requires structural information . RESULTS: The crystal structures of oxidized and reduced OYE at 2 A resolution reveal an alpha/beta-barrel topology clearly related to trimethylamine dehydrogenase . Complexes of OYE with p-hydroxybenzaldehyde, beta-estradiol, and an NADPH analog show all three binding at a common site, stacked on the flavin . The putative NADPH binding mode is novel as it involves primary recognition of the nicotinamide mononucleotide portion . CONCLUSIONS: This work shows that the striking spectral changes seen upon phenol binding are due to close physical association of the flavin and phenolate . It also identifies the structural class of OYE and suggests that if NADPH is its true substrate, then OYE has adopted NADPH dependence during evolution. Eur J Biochem, 1994 Nov 1, 225(3), 985 - 93 Structure of the Drosophila melanogaster glutathione-dependent formaldehyde dehydrogenase/octanol dehydrogenase gene (class III alcohol dehydrogenase) . Evolutionary pathway of the alcohol dehydrogenase genes; Luque T et al.; The glutathione-dependent formaldehyde dehydrogenase gene (gfd) of Drosophila melanogaster encodes an enzyme that is active toward S-hydroxymethylglutathione, an adduct of formaldehyde with glutathione, and also with long-chain primary alcohols, both properties typical of class III alcohol dehydrogenases, gfd hybridizes at the 86D division of the third chromosome, in agreement with the known location of the Drosophila octanol dehydrogenase gene (odh), gfd/odh was isolated from a lambda EMBL-4 genomic library and consists of three exons (with coding segments of 21, 90 and 1029 bp) and two introns (69 bp and 70 bp, respectively) . The introns are small in size like the Drosophila interrupting sequences and are located at the 5' end of the coding region . Comparisons with the homologous genes of Saccharomyces, Candida and humans provide information on the evolution of the class III alcohol dehydrogenases . Moreover, results from analysis of exon/intron distributions in eleven dehydrogenases are compatible with the hypothesis of intron loss accounting for aspects of the present structure of these genes. Bioessays, 1994 Aug, 16(8), 557 - 64 Roadblocks and detours during DNA replication: mechanisms of mutagenesis in mammalian cells; Naegeli H; Mutations in specific genes result in birth defects, cancer, inherited diseases or lethality . The frequency with which DNA damage is converted to mutations increases dramatically when the cellular genome is replicated . Although DNA damage poses special problems to the fidelity of DNA replication, efficient mechanisms exist in mammalian cells which function to replicate their genome despite the presence of many damaged sites . These mechanisms operate in either error-prone or error-free modes of DNA synthesis, and frequently involve DNA strand-pairing reactions . Genetic studies in yeast and other eukaryotes suggest that replication through DNA damage is highly regulated and catalysed by complex biochemical machineries composed of many specialized gene products . Knowledge of the molecular details by which such factors facilitate the replication of damaged DNA in mammalian cells should reveal basic rules about how DNA damage induces mutagenesis and carcinogenesis. Gene, 1994 Jun 10, 143(2), 165 - 70 Cloning and growth-regulated expression of the gene encoding the hepatitis B virus middle surface antigen in Yarrowia lipolytica; Hamsa PV et al.; Expression of the gene encoding the hepatitis B virus middle surface antigen (pre-HBsAg) in the yeast Yarrowia lipolytica has been studied . The preS2-HBsAg gene was expressed from the alkaline extracellular protease-encoding gene (XPR2) promoter . In the fusion construct, the membrane-spanning 'a' domain of preS2-HBsAg has been replaced by the leader peptide and the proI region of the alkaline protease, thus eliminating the epitope responsible for the immune escape mechanism . Expression has been found to be growth-stage dependent with the highest protein accumulation during the stationary phase, accounting for around 2.35% of the total soluble intracellular proteins . The produced protein was assembled into Dane particles and was immunogenic in mice . The expression vector was found to be stable for at least 100 generations under non-selective conditions. Development, 1994 Jun, 120(6), 1503 - 15 Developmental control of a G1-S transcriptional program in Drosophila; Duronio RJ et al.; We have defined a coordinate program of transcription of S-phase genes (DNA polymerase alpha, PCNA and the two ribonucleotide reductase subunits) that can be induced by the G1 cyclin, cyclin E . In Drosophila embryos, this program drives an intricate spatial and temporal pattern of gene expression that perfectly parallels the embryonic program of S-phase control . This dynamic pattern of expression is not disrupted by a mutation, string, that blocks the cell cycle . Thus, the transcriptional program is not a secondary consequence of cell cycle progression . We suggest that developmental signals control this transcriptional program and that its activation either directly or indirectly drives transition from G1 to S phase in the stereotyped embryonic pattern. J Bacteriol, 1994 May, 176(9), 2477 - 82 Ylt1, a highly repetitive retrotransposon in the genome of the dimorphic fungus Yarrowia lipolytica; Schmid-Berger N et al.; A highly repetitive composite element, Ylt1, was detected in the genome of the dimorphic fungus Yarrowia lipolytica . Ylt1 resembles retrotransposons found in other eukaryotes . It is about 9.4 kb long and can transpose in the genome . The Ylt1 element is bounded by a long terminal repeat (LTR), the zeta element . Several copies of zeta were isolated and sequenced . The sequence of this element is well conserved . It is 714 bp long and is bounded by nucleotides 5'-TG...CA-3', which are part of a short inverted repeat, a feature conserved in the LTRs of retroviruses and retrotransposons . Sequence analysis revealed motifs commonly found in LTR elements, like signals for the start and termination of transcription . The zeta element exists as part of retrotransposon Ylt1, as well as a solo element in the genome . Ylt1 and solo zeta elements are flanked by a 4-bp directly repeated genomic sequence . The copy numbers of Ylt1 and solo zeta are dependent on the strain examined, but at least 35 copies of the composite Ylt1 element and more than 30 copies of the solo zeta element per haploid genome have been observed. Biochem J, 1994 Apr 15, 299 ( Pt 2), 335 - 40 A meiotic DNA polymerase from a mushroom, Agaricus bisporus; Takami K et al.; A meiotic DNA polymerase {DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7}, which likely has a role in meiotic DNA repair, was isolated from a mushroom, Agaricus bisporus . The purified fraction displays three bands in SDS/PAGE, at molecular masses of 72 kDa, 65 kDa and 36 kDa . Optimal activity is at pH 7.0-8.0 in the presence of 5 mM Mg2+ and 50 mM KCl and at 28-30 degrees C, which is the temperature for meiosis . This enzyme is resistant to N-ethylmaleimide and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, suggesting that it is a beta-like DNA polymerase . These characteristics are similar to those of Coprinus DNA polymerase beta {Sakaguchi and Lu (1982) Mol . Cell . Biol . 2, 752-757} . In Western-blot analysis, the antiserum against the Coprinus polymerase reacts only with the 65 kDa band, which coincides with the molecular mass of the Coprinus polymerase . Western-blot analysis also showed that the antiserum could react with crude extracts not only from the Agaricales family, to which Agaricus and Coprinus belong, but also from different mushroom families and Saccharomyces . The Agaricus polymerase activity can be found only in the meiotic-cell-rich fraction, but the enzyme is also present in the somatic cells in an inactive state. Nature, 1994 Mar 17, 368(6468), 258 - 61 Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer; Bronner CE et al.; The human DNA mismatch repair gene homologue hMSH2, on chromosome 2p is involved in hereditary non-polyposis colon cancer (HNPCC) . On the basis of linkage data, a second HNPCC locus was assigned to chromosome 3p21-23 (ref . 3) . Here we report that a human gene encoding a protein, hMLH1 (human MutL homologue), homologous to the bacterial DNA mismatch repair protein MutL, is located on human chromosome 3p21.3-23 . We propose that hMLH1 is the HNPCC gene located on 3p because of the similarity of the hMLH1 gene product to the yeast DNA mismatch repair protein, MLH1, the coincident location of the hMLH1 gene and the HNPCC locus on chromosome 3, and hMLH1 missense mutations in affected individuals from a chromosome 3-linked HNPCC family. Bull Group Int Rech Sci Stomatol Odontol, 1994 Mar-Jun, 37(1-2), 13 - 7 "Hanseniaspora uvarum" the ultrastructural morphology of a rare ascomycete, isolated from oral thrush; Emmanouil-Nikoloussi E et al.; Superficial fungal infections, including oral thrush, often affect aged full denture wearers and many individuals over 65 years old . The aim of this study was to examine the ultrastructural morphology of a very rare yeast, named Hanseniaspora uvarum/guillermondi, member of the Ascomycetes family, whose pathogenesis and behaviour is not widely known . The yeast was isolated from whitish lesions of the buccal mucosa of an 70 years old woman . The specimen was collected with a mouth swab and cultured in Sabourauds-Dextrose agar . The identification of the organism was performed on the Api 20C Aux system . The yeast colonies, after fixation in glutaraldehyde 3% for 1 hour were immersed in OsO4 1% solution for 1 hour and were "in tissue" stained with uranyl acetate . Ultrathin sections, were observed with TEM Jeol C x 100 . Our ultrastructural observations showed that this yeast had a thick cell wall in which the outer surface appeared fuzzy . In some yeasts we observed multilayered intracytoplasmic membrane a figure which is not described as far as we know in any yeast . Many vacuoles were frequently observed in the cytoplasm and especially in the center of the oval shaped cells . Bilateral budding which form ascospores is identical for the morphology of this yeast. J Nat Prod, 1994 Jan, 57(1), 68 - 73 Isolation of bioactive and other oxoaporphine alkaloids from two annonaceous plants, Xylopia aethiopica and Miliusa cf . banacea; Harrigan GG et al.; The oxoaporphine alkaloids oxophoebine {1} and liriodenine {2} have been isolated from Xylopia aethiopica (Annonaceae) . Both showed selective toxicity against DNA repair and recombination deficient mutants of the yeast Saccharomyces cerevisae . Three related but inactive compounds, oxoglaucine {3}, O-methylmoschatoline {4}, and lysicamine {5}, were also isolated from this plant . Selective toxicity was also observed for 10-methoxyliriodenine (lauterine) {6} and 10-hydroxyliriodenine {7}, two oxoaporphine alkaloids isolated from Miliusa cf . banacea (Annonaceae) . The structure of 10-hydroxyliriodenine {7}, a novel oxoaporphine, was determined by spectroscopic methods and chemical conversion to compound 6 . The role of the bioactive oxoaporphine alkaloids as DNA topoisomerase inhibitors is discussed. Can J Microbiol, 1994 Jan, 40(1), 54 - 9 Biochemical and physiological characteristics of Yarrowia lipolytica strains in relation to isolation source; Sinigaglia M et al.; The physiological characteristics and fatty acid composition of 40 strains of Yarrowia lipolytica isolated from irradiated poultry meat, commercial chilled foods, the superficial water of lagoons of an Italian river delta, and commercial light butter were compared . Discriminant analysis of the results permitted the 40 strains to be classified into four groups according to their isolation source . The lag phase length at 3, 5, and 7 degrees C, the proteolytic and lipolytic activities, and the growth on sorbitol, gluconate, and N-acetylglucosamine, as well as the relative percentages of C12:0, C15:0, C17:1, C18:1, C18:2, the unsaturation level, and the percentage of total odd chain fatty acids were the characteristics exhibiting the highest discriminatory power . These results indicate that the isolates are well adapted and significant biochemical characteristics may account for the success of individual strains in their original habitat. Antonie Van Leeuwenhoek, 1994, 65(2), 107 - 9 Medium-induced fragility of Schwanniomyces; Johnson BF et al.; Schwanniomyces occidentalis has attracted interest because of its ability to metabolize starch and similar complex carbohydrates . Studies have been undertaken, mostly using defined media, to ascertain conditions for optimal production and secretion of hydrolytic enzymes . Here we demonstrate the fragility of Schw . occidentalis in many defined media . We especially examined viability in YNB (Yeast Nitrogen Base) plus 1% glucose . Without phosphate supplementation, viability was routinely very low at stationary phase (usually less than 37%), whereas viability of stationary-phase cultures in phosphate-supplemented YNB usually exceeded 97% . The negative implications of having many, presumably permeabilized, dead cells present in assays for secretion of enzymes by living yeast cells are discussed. FEBS Lett, 1993 Oct 25, 333(1-2), 39 - 43 The primary structure of carboxypeptidase S1 from Penicillium janthinellum; Svendsen I et al.; The complete amino acid sequence of carboxypeptidase S1 from Penicillium janthinellium has been determined by N-terminal sequencing of the reduced and vinylpyridinated protein and of peptides obtained by cleaved with cyanogen bromide, iodosobenzoic acid, hydroxylamine, endoproteinase LysC, endoproteinase AspN and Glu-specific proteinase from B . licheniformis . The enzyme consists of a single peptide chain of 433 amino acid residues and contains 9 half-cystine residues and one glycosylated asparagine residue . A comparison to other carboxypeptidases shows that the enzyme is homologous to carboxypeptidase-Y and carboxypeptidase-MIII from malt . Specificity and binding of substrates are discussed from a three-dimensional model based on the known structure of carboxypeptidase-Y from Saccharomyces cereviciae and carboxypeptidase II from wheat. Environ Health Perspect, 1993 Oct, 101 Suppl 3, 5 - 9 Development of screening tests for aneuploidy induction by environmental pollutants; Adler ID et al.; When legally required mutagenicity testing of chemicals is undertaken, the important genetic end point of aneuploidy is not included because validated test methods are lacking . Therefore, the Commission of the European Communities (CEC) has funded a research program to develop and validate tests for aneuploidy induction . Ten chemicals, selected on the basis of their ability to interact with cell organelles relevant for aneuploidy induction, were tested in 11 laboratories . The assays ranged from in vitro tubulin assembly studies to in vivo germ-cell tests . The results allow several conclusions: a) Fungal aneuploidy tests are not capable of detecting inhibitors of mammalian tubulin polymerization such as colchicine and vinblastine . Therefore, they will not play a role in screening for aneuploidy but are of value for studying the relationship between induced aneuploidy and recombination . b) Chemicals that induce aneuploidy in mammalian germ cells are readily detected in the in vitro mammalian cell systems . Some chemicals such as thiabendazole and thimerosal induce aneuploidy in vitro but do not appear to be very effective in vivo . c) Cell division aberrations induced in mammalian cells in vitro seem to be predictive for aneuploidy induction in the same cell type . Likewise, c-mitotic effects and cell cycle delay in vivo in mitotic and meiotic cells correlate with aneuploidy induction in the respective tissue . A second CEC Aneuploidy Program has started recently to refine the most promising test protocols, to provide understanding of variety of mechanisms by which chemicals induce aneuploidy, and to establish a data base for aneugens among environmental pollutants. Enzyme Microb Technol, 1993 Aug, 15(8), 699 - 702 Rapid release of protoplasts from Eremothecium ashbyii in comparison with Trichoderma reesei and Penicillium chrysogenum using novozyme and funcelase; Lakshmi BR et al.; Protoplast release in Eremothecium ashbyii, Trichoderma reesei, and Penicillium chrysogenum was achieved using commercially available enzymes, Novozyme 234 and Funcelase . A rapid release of protoplasts was observed in E . ashbyii, yielding nearly 4.0 x 10(7) protoplasts ml-1 in 10-35 min . The regeneration frequency of protoplasts from T . reesei, P . chrysogenum, and E . ashbyii using Funcelase was 51.77, 28.32, and 7.64%, respectively, and was higher in comparison with Novozyme-derived protoplasts. Curr Genet, 1993 Jul-Aug, 24(1-2), 75 - 83 Molecular structure of the SWA2 gene encoding an AMY1-related alpha-amylase from Schwanniomyces occidentalis; Claros MG et al.; A 2.1-kb DNA fragment containing the SWA2 gene determining an alpha-amylase from Schwanniomyces occidentalis has been sequenced . It contains an open reading frame of 1521 bp which has the potential to encode a 507 amino-acid protein of M(r) 55966 . Its deduced amino-acid sequence shows significant similarities to the sequence of other studied alpha-amylases . These similarities identify a consensus sequence, F(LIV)(ED)NHD, which is shared in addition by most maltases, invertases and glucoamylases. Biochemistry, 1993 Jun 22, 32(24), 6220 - 8 Parallel and antiparallel G-DNA structures from a complex telomeric sequence; Venczel EA et al.; We investigated the formation in vitro of higher order structures by a DNA oligomer containing the terminal motif TGTG3TGTGTGTG3, derived from the Saccharomyces telomeric consensus, in order to (a) understand why certain cations favor the formation of parallel-stranded (G4 and G8) G-DNA structures, while others favor foldback, antiparallel structures (G'2) and (b) probe the structures of G-DNAs formed by this telomeric sequence, which is more complex than its well-studied counterparts from the protozoans oxytricha and tetrahymena . We find that dramatic switches in the formation of G4 versus G'2 structures occur in solutions of not only the group Ia cations, Li(+)-Cs+, but also in those of the group IIa cations, Mg(2+)-Ba2+ . These data and the temperature-dependent formation and destruction of the different structures lend support to the kinetic scheme of Sen and Gilbert (1990), by which rapidly forming G'2 structures accumulate in highly stabilizing potassium (and strontium) solutions at the expense of the thermodynamically more stable G4 structures . Both the G4 and the G'2 complexes formed by the Saccharomyces sequence show novel structural features . Protection and interference experiments with dimethyl sulfate and potassium permanganate reveal that the core of alternating thymines and guanines within the telomeric motif plays a critical role in the stabilization of the parallel G4 structure, but not of the antiparallel G'2 . Very likely, in the G4 complex, this GT core forms a novel higher order arrangement of alternating G and T quartets, the latter possibly comparable to the U quartets described by Cheong and Moore (1992) in their NMR study of the higher order structure formed by rUG4U. Biochemistry, 1993 Jun 22, 32(24), 6165 - 70 Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution; Dyda F et al.; The crystal structure of brewers' yeast pyruvate decarboxylase, a thiamin diphosphate dependent alpha-keto acid decarboxylase, has been determined to 2.4-A resolution . The homotetrameric assembly contains two dimers, exhibiting strong intermonomer interactions within each dimer but more limited ones between dimers . Each monomeric subunit is partitioned into three structural domains, all folding according to a mixed alpha/beta motif . Two of these domains are associated with cofactor binding, while the other is associated with substrate activation . The catalytic centers containing both thiamin diphosphate and Mg(II) are located deep in the intermonomer interface within each dimer . Amino acids important in cofactor binding and likely to participate in catalysis and substrate activation are identified. FEMS Microbiol Lett, 1993 May 1, 109(1), 45 - 8 Phosphatase activity during growth of Yarrowia lipolytica; Galabova D et al.; The yeast Yarrowia lipolytica produces four patterns of phosphatase activity during growth in the presence or absence of inorganic phosphate in the medium . Activities had pH optima at 4.2, 5.8, about pH 6.5 and pH 9.0 . The level of all four phosphatase activities depended on the presence of inorganic phosphate in the medium. Curr Opin Cell Biol, 1993 Apr, 5(2), 180 - 6 Activation of the various cyclin/cdc2 protein kinases; Solomon MJ; Recent research has led to the near culmination of the biochemical confirmation of the most basic genetic predictions about how p34cdc2 activity is controlled . The field is now moving from a biochemical dissection of the machinery to understanding how the system is regulated . The discovery of numerous, highly related protein kinase complexes that control other cell cycle events will test the generality of this paradigm. Medicine (Baltimore), 1993 Mar, 72(2), 78 - 89 The spectrum of non-Candida fungal infections following bone marrow transplantation; Morrison VA et al.; We evaluated a consecutive series of patients who underwent bone marrow transplantation (BMT) at a single institution between 1974 and 1989 for the occurrence of a non-Candida fungal infection in the first 180 days after BMT . Of the 1186 patients, 129 (11%) patients developed a total of 138 significant non-Candida fungal infections in this period . Eight patients had multiple distinct infections . The most common isolate was Aspergillus spp . (n = 97), followed by Fusarium (n = 10), and Alternaria (n = 6) . The 4 clinical subtypes of infections were minor skin or soft-tissue infections (n = 7), infections of a single organ or site (n = 61), disseminated fungal infection (n = 58), and isolated fungemia (n = 12) . The respiratory tract was involved in 95% of single organ or site infections, and 84% of disseminated infections . Outcome was poor, with only 18% of patients surviving . The cause of death was directly related to the non-Candida fungal infection in 66% of patients who died . Mortality rates were significantly higher in patients with either single-organ or site infections (41%) or disseminated infections (83%) . The cause-specific mortality rate was greatest following infections with Aspergillus, Chrysosporium, Fusarium, Mucor, or Scopulariopsis, in which there was a high potential for invasive disease and disseminated infection . In contrast, the cause-specific mortality rate was lowest in infections which were either isolated fungemia or were localized and amenable to surgical debridement, most often seen with those infections caused by Acremonium, Alternaria, Penicillium, and Saccharomyces . The spectrum of clinical infections caused by these uncommon non-Candida fungal isolates both in our series and in the literature is reviewed . These unusual opportunistic fungal isolates are now gaining recognition in immunosuppressed patients such as the BMT population, and have a significant impact on patient outcome . Effective therapy of non-Candida fungal infections remains difficult . Early aggressive surgical debridement appears to be important in control of localized invasive infections . Prolonged therapy with amphotericin B is the standard of care, although the role of the newer antifungal agents is not yet well-defined . Ancillary roles may also be provided by granulocyte transfusions and the colony-stimulating factors. J Gen Microbiol, 1993 Mar, 139 ( Pt 3), 485 - 93 The role of polyamine metabolism in dimorphism of Yarrowia lipolytica; Guevara-Olvera L et al.; We have devised a convenient procedure to induce the yeast-to-mycelium transition of Yarrowia lipolytica in conditions which avoid the occurrence of the reverse process during the period of study . Yeast cells in late exponential phase were resuspended in water and cooled down to 4 degrees C for at least 15 min, then heat-shocked by inoculation into a pre-warmed (30 degrees C) medium containing N-acetyl-D-glucosamine . Under these conditions, yeast cells developed into large branching filaments which continued elongating for more than 24 h . Further, ornithine decarboxylase (ODC) activity and polyamine cell pools increased compared to those of cells maintained in glucose medium, which continued yeast-like growth . Addition of ODC inhibitors blocked mycelial development, but only if added during a critical initial period after which they had no effect . At effective concentrations, ODC inhibitors had no significant effect on cell growth . Comparative studies of intact and permeabilized cells suggest that this selective effect is probably due to the location of ODC in more than one cell compartment, one of them being inaccessible to the drugs . Blocking of the morphological transition by ODC inhibitors was specifically reversed by putrescine, and by growing the cells in the presence of 5-azacytidine . It is suggested that the effect of the latter compound is related to its capacity to inhibit DNA methylation, indicating a relationship between polyamines and DNA methylation at the onset of the differentiation process. Antonie Van Leeuwenhoek, 1993 Feb, 63(2), 111 - 23 The distribution and taxonomic value of fatty acids and eicosanoids in the Lipomycetaceae and Dipodascaceae; Botha A et al.; Using radioimmunoassay, blood platelet aggregation studies and GC-MS the existence of prostaglandins in the endomycetalean yeast Dipodascopsis uninucleata was confirmed by our group . These findings triggered the search for similar eicosanoids in the rest of the Endomycetales . We commenced by scanning for the easily detectable precursors of eicosanoids, linoleic- and linolenic acid . We selected two families (i.e . Lipomycetaceae and Dipodascaceae), both producing these precursors, for further investigation . Representative strains of the two families were tested for their ability to grow in the presence of 1 mM aspirin, a specific inhibitor of prostaglandin biosynthesis . In contrast to the lipomycetaceous species the dipodascaceous species were insensitive to this drug . These results were verified when representative strains of both families were investigated for their ability to produce eicosanoids from externally fed radio-labeled arachidonic acid along an aspirin sensitive pathway . Thin layer chromatography of culture extracts, followed by autoradiography, showed that while none of the Dipodascaceae produced aspirin sensitive arachidonic acid metabolites, the members of the Lipomycetaceae tested positive for these metabolites . These findings supported the separation of the lipomycetaceous yeast Dipodascopsis from the Dipodascaceae . The findings also correlate with the delimitation of these yeasts in two families (i.e . Dipodascaceae and Lipomycetaceae) . Further investigation indicated that prostaglandin production by the genus Dipodascopsis is mainly associated with ascosporogenesis . Thin layer chromatography of cell extracts from Dipodascopsis tothii, followed by scintillation counting, indicated the presence of PGF2 alpha and PGE2 during ascosporogenesis. Nucleic Acids Res, 1993 Jan 25, 21(2), 239 - 43 Molecular characterisation and stage-specific expression of proliferating cell nuclear antigen (PCNA) from the malarial parasite, Plasmodium falciparum; Kilbey BJ et al.; The gene encoding the malarial homologue of proliferating cell nuclear antigen, PCNA, has been identified and characterised . It is located on chromosome 13 . The coding sequence of 825 nucleotides predicts a protein of 30,586 Da . There are no introns and northern analysis reveals a transcript of approximately 1.6kb . The conserved residues which characterise the PCNAs of human, Drosophila, Saccharomyces and Xenopus are present in PfPCNA but the overall identity of PfPCNA with human and yeast PCNAs is low; 34% and 31% respectively . PfPCNA is longer than the PCNAs of these other species by about 16 amino acids, most of which are present in a block near the carboxy terminus . Antibodies against a purified PfPCNA-glutathione-S-transferase fusion protein recognise a single band in western blots of parasite extracts at 32kDa . The same antiserum has been used to demonstrate that the expression of PfPCNA is regulated during the intraerythrocytic development of the parasite . Expression increases dramatically in late trophozoites and is maintained during the subsequent nuclear divisions which produce schizonts. FEBS Lett, 1992 Dec 14, 314(2), 195 - 8 Gamma-COP, a coat subunit of non-clathrin-coated vesicles with homology to Sec21p; Stenbeck G et al.; Constitutive secretory transport in eukaryotes is likely to be mediated by non-clathrin-coated vesicles, which have been isolated and characterized {(1989) Cell 58, 329-336; (1991) Nature 349, 215-220} . They contain a set of coat proteins (COPs) which are also likely to exist in a preformed cytosolic complex named coatomer {(1991) Nature 349, 248-250} . From peptide sequence and cDNA structure comparisons evidence is presented that one of the subunits of coatomer, gamma-COP, is a true constituent of non-clathrin-coated vesicles, and that gamma-COP is related to sec 21, a secretory mutant of the yeast Saccharomyces cervisiae. FEBS Lett, 1992 Dec 7, 314(1), 101 - 3 Correlation of cofactor binding and the quaternary structure of pyruvate decarboxylase as revealed by 31P NMR spectroscopy; Hubner G et al.; The pH dependence of the quaternary structure of pyruvate decarboxylase (EC 4.1.1.1) has recently been discovered {(1990) FEBS Lett . 266, 17-20; (1992) Biochemistry (in press)} . In the present study we have investigated the change in quaternary structure by observing the binding of the cofactor, thiamine pyrophosphate, using 31P NMR spectroscopy . The dissociation of the native tetramers into dimers when increasing the pH coincides with a weaker binding of the cofactor and loss of enzyme activity . The results provide further evidence that thiamine pyrophosphate is bound primarily via the beta-phosphate moiety . In addition, a phosphoserine has been discovered in two of the four subunits. Antonie Van Leeuwenhoek, 1992 Nov, 62(4), 251 - 9 Evidence for, and taxonomic value of, an arachidonic acid cascade in the Lipomycetaceae; Kock JL et al.; By using specific inhibitors of the lipoxygenase and cyclo-oxygenase pathways, arachidonic acid metabolites with similar sensitivities towards these inhibitors as in humans, were detected in Dipodascopsis uninucleata . The taxonomic value of aspirin sensitive arachidonic acid metabolites in the Lipomycetaceae was next assessed . No metabolites of which the production is inhibited by aspirin were detected in strains representing the following species: Lipomyces starkeyi, Lipomyces kononenkoae, Lipomyces tetrasporus, Myxozyma melibiosi, Myxozyma mucilagina, Myxozyma kluyveri, Waltomyces lipofer, Zygozyma oligophaga and Zygozyma arxii . The detection of such aspirin sensitive arachidonic acid metabolites in representative strains of Lipomyces anomalus and the genus Dipodascopsis, emphasises the isolated position of these taxa in the genus Lipomyces and the family Lipomycetaceae, respectively . Finally using long chain fatty acid analyses, electrophoretic karyotyping and other phenotypic characters, a phylogenetic scheme is proposed for some genera in the Lipomycetaceae. Antonie Van Leeuwenhoek, 1992 Nov, 62(4), 247 - 50 The Lipomycetaceae, a model family for phylogenetic studies; van der Walt JP; The Lipomycetaceae (Endomycetales) are known from the genera Dipodascopsis, Lipomyces and Zygozyma with budding anamorphic states in Myxozyma . The family is easily recognized culturally and physiologically but is phenotypically and ecologically extremely diverse . This natural taxon is phylogenetically distinct from the Saccharomycetaceae, but probably related to the Dipodascaceae . The possible evolution of the lipomycetaceous anamorphs is discussed. Infect Immun, 1992 Oct, 60(10), 4140 - 5 Fungus-specific translation elongation factor 3 gene present in Pneumocystis carinii; Ypma-Wong MF et al.; Historically, Pneumocystis carinii pneumonia has been the most frequent cause of morbidity and mortality in patients with AIDS . Antiprotozoan drugs are effective in the treatment and prophylaxis of P . carinii pneumonia, which lends credence to the widely held view that P . carinii is a protozoan . However, recent genetic evidence suggests that P . carinii should be classified as a fungus . Translation elongation factor 3 (EF-3) is an essential, soluble translation component which is unique to fungal protein synthesis and is not required for protein synthesis in other eukaryotes . We have identified and isolated a gene for EF-3 from P . carinii, adding more evidence for this organism's assignment as a fungus. Antonie Van Leeuwenhoek, 1992 Aug, 62(1-2), 15 - 24 Topoisomerase II: its functions and phosphorylation; Gasser SM et al.; The gene encoding topoisomerase II in yeast is unique and essential, required for both mitotic and meiotic proliferation . The use of temperature-sensitive mutants in topoisomerase II have demonstrated roles in the relaxation of tortional stress, reduction of recombination rates, and in the separation of sister chromatids after replication . In vertebrate cells, topoisomerase II was shown to be the most abundant component of the metaphase chromosomal scaffold, and has been shown to play a role in chromosome condensation in vitro . The cell cycle control of chromosome condensation may well require phosphorylation of topoisomerase II, since the enzyme is more highly phosphorylated in metaphase than in G1 . Recent studies have identified casein kinase II as the major enzyme phosphorylating topoisomerase II in intact yeast cells . The target sites of CKII are exclusively in the C-terminal 400 amino acids of topoisomerase II, the region that is most divergent among the eukaryotic type II enzymes and which is absent in the bacterial gyrase homologues. Biochim Biophys Acta, 1992 May 21, 1106(2), 251 - 6 Interfacial pH modulation of membrane protein function in vivo . Effect of anionic phospholipids; Calderon V et al.; In yeast cells, the magnitude of the membrane surface potential (phi) is determined to a large extent by the relative amount of anionic phospholipids (Cerbon and Calderon (1990) Biochim . Biophys . Acta 1028, 261-267) . When a significant surface potential exists, the pH at the membrane surface (interfacial pH) will be different to that in the bulk suspending medium . We now report that: (1) In cells with higher phi (phosphatidylinositol-rich cells (PI-rich) and phosphatidylserine-rich cells (PS-rich) a 10-times lower proton concentration in the bulk was enough to achieve the maximum transport activity of H(+)-linked transport systems when compared to normal cells . (2) When the phi was reduced by increasing the concentration of cations in the medium, more protons were required to achieve maximum transport, that is, the pH activity curves shifted downwards to a more acidic pH . (3) The magnitude of the downward pH shift was around 2.5-times higher for the more charged membranes . (4) Around 10-times more KCl than MgCl2 was necessary to give an equivalent pH shift, in agreement with their capacity to reduce the phi of artificial bilayers . The interfacial pH calculated from the values of phi indicates that it was 0.4 pH units lower in the anionic phospholipid rich cells as compared to normal cells . The results indicate that membrane surface potential may explain the complex relationship between pH, ionic strength and membrane protein function . Maximum transport activities were found for glutamate at interfacial pH of 4.2-4.8 and were inhibited at interfacial pH = 3.2-3.4, suggesting that surface groups of the carrier proteins with pK values in the region 3.8-4.2 (aspartyl and glutamyl) are involved in binding and/or release of charged substrates. J Cell Sci, 1992 Apr, 101 ( Pt 4), 773 - 84 A monoclonal antibody recognizing nuclear matrix-associated nuclear bodies; Stuurman N et al.; We have isolated a monoclonal antibody, 5E10, that labels discrete spots in the interphase nucleus . By immunoblotting mAb 5E10 recognized predominantly a 126 kDa polypeptide with an isoelectric point of 5.5 . Indirect immunofluorescence showed that mAb 5E10 labeled spots in many cell lines and tissues from rat or human origin, but not in cells from mouse, chicken, African green monkey, or the lower eukaryotes Saccharomyces and Dictyostelium . In the human bladder carcinoma cell line T24 the number of nuclear spots were found to be 21 +/- 10 (n = 132) . In many cells spots were found also in the cytoplasm . In a small fraction of T24 cells the mAb revealed thread-like structures in addition to spots . Throughout mitosis the antigen was found to be clustered in the cytoplasm, not associated with metaphase chromosomes . The spherical structures that contain the antigen were tightly bound to the nuclear matrix . Immunogold labeling with mAb 5E10 showed that the antigen is localized in 0.3 microns diameter spherical, electron-dense structures, reminiscent of nuclear bodies . Double-labeling experiments showed that these spots do not colocalize with U1 snRNPs and centromeres . The spots did colocalize with nuclear speckles recognized by a primary biliary cirrhosis autoimmune serum, which is thought to recognize nuclear bodies . On the basis of these observations we conclude that mAb 5E10 recognizes discrete nuclear substructures, most likely nuclear bodies. FEBS Lett, 1992 Mar 30, 300(2), 119 - 22 Cloning and sequencing of cDNA encoding the putative insect plasma membrane V-ATPase subunit A; Graf R et al.; For the first time a cDNA encoding subunit A of an invertebrate V-ATPase has been sequenced . The cDNA library was prepared from larval midgut of the tobacco hornworm, Manduca sexta, and screened with monoclonal antibodies to the midgut plasma membrane subunit A . From the cDNA sequence the insect subunit A is predicted to consist of 617 amino acids with a relative molecular mass of 68,162 . The predicted primary structure is similar to that of the published eukaryotic subunit A proteins (Bos, Daucus, Saccharomyces and Neurospora); it most closely resembles the bovine amino acid sequences with which it has an 83% sequence identity. Vopr Pitan, 1992 Mar-Apr, (2), 38 - 42 {Adaptation of rats to unusual protein products during transition to definitive nutrition}; Gorodina SM et al.; The authors studied the adaptation of initial and final links of the proteolytic chain, and intermediate nitrogen metabolism, to unusual products--protein concentrate from yeast-saccharomyces and anchovy . A number of enzymes in the organs-producers were found to be activated due to intensification of a limited proteolytic reaction . It was established that the changes in the functioning of the proteolytic stage of the digestive-transport conveyor led to shifts in the amino-acid pool in the blood serum of experimental animals. J Biol Chem, 1992 Jan 25, 267(3), 1932 - 7 A mutant of 7SL RNA in Yarrowia lipolytica affecting the synthesis of a secreted protein; He F et al.; The yeast Yarrowia lipolytica contains two genes, SCR1 and SCR2, encoding a 7SL RNA associated with a signal recognition particle (SRP) . To study 7SL RNA function in vivo we have systematically substituted the two conserved nucleotides G128 and A130 in loop 1 of SCR2-encoded 7SL RNA . All single mutations in either nucleotide have no effect . All double mutations are lethal except for one which results in temperature-sensitive growth . We have studied the synthesis and secretion of an alkaline extracellular protease (AEP) in both wild-type and temperature-sensitive mutant strains . Pulse-chase labeling and immunoprecipitation of this protein show that: 1) total protein synthesis is not affected in the mutant strain; 2) levels of AEP precursors in the mutant strain are 60% less than in the wild-type strain; 3) for both strains there is no accumulation of the AEP precursors in the cytoplasm; 4) the kinetics of secretion is similar . These results suggest that mutated SRP is deficient in membrane targeting but still performs translational arrest . This is consistent with the functions of SRP observed in vitro and represents the first demonstration of such roles in vivo. Science, 1992 Jan 17, 255(5042), 327 - 30 Cyclic 2',3'-phosphates and nontemplated nucleotides at the 3' end of spliceosomal U6 small nuclear RNA's; Lund E et al.; Spliceosomal U6 small nuclear RNA (U6 RNA) in species as diverse as man, frog, fruitfly, and soybean have at their 3' ends a cyclic 2',3'-phosphate (greater than p) apparently derived from uridylic acid residues that were added post-transcriptionally . The 3' ends of U6 RNA's from various sources may be processed in different ways, or to different extents, depending on the organism or stage of development . The presence of a greater than p terminus on U6 RNA may influence the activity of U6 RNA either directly during splicing or indirectly by ensuring that the RNA has a defined length or proper conformation (or both). Plant Mol Biol, 1992 Jan, 18(1), 33 - 45 Structure of a rice beta-glucanase gene regulated by ethylene, cytokinin, wounding, salicylic acid and fungal elicitors; Simmons CR et al.; A rice beta-glucanase gene was sequenced and its expression analyzed at the level of mRNA accumulation . This gene (Gns1) is expressed at relatively low levels in germinating seeds, shoots, leaves, panicles and callus, but it is expressed at higher levels in roots . Expression in the roots appears to be constitutive . Shoots express Gns1 at much higher levels when treated with ethylene, cytokinin, salicylic acid, and fungal elicitors derived from the pathogen Sclerotium oryzae or from the non-pathogen Saccharomyces cereviseae . Shoots also express Gns1 at higher levels in response to wounding . Expression in the shoots is not significantly affected by auxin, gibberellic acid or abscisic acid . The beta-glucanase shows 82% amino acid similarity to the barley 1,3;1,4-beta-D-glucanases, and from hybridization studies it is the beta-glucanase gene in the rice genome closest to the barley 1,3;1,4-beta-glucanase EI gene . The mature peptide has a calculated molecular mass of 32 kDa . The gene has a large 3145 bp intron in the codon for the 25th amino acid of the signal peptide . The gene exhibits a very strong codon bias of 99% G + C in the third position of the codon in the mature peptide coding region, but only 61% G + C in the signal peptide region. J Basic Microbiol, 1992, 32(1), 57 - 63 Transport and hydrolysis of maltose by Schwanniomyces castellii; Violle P et al.; Hydrolysis and transport of maltose into Schwanniomyces castellii was studied under aerobic and anaerobic conditions . Amylase and glucosidase were not synthetized in presence of maltose in anaerobic conditions . Maltose permease was synthesized in anaerobiosis and its functioning is not inhibited . Glucose strongly repressed induction . The half-saturation constant for uptake of maltose was 0.06 mM . The rate of uptake of maltose was decreased by 2,4-dinitrophenol, antimycin or sodium azide . The significance of these results in relation to the Kluyver effect is discussed. J Protozool, 1991 Nov-Dec, 38(6), 66S - 68S PCR amplification of DNA sequences from the transcription factor IID and cation transporting ATPase genes in Pneumocystis carinii; Meade JC et al.; Oligonucleotide primers were used to amplify DNA sequences from a plasma membrane cation transporting ATPase gene and a transcription factor IID (TFIID) gene from Pneumocystis carinii genomic DNA . The entire P . carinii ATPase gene was cloned from a genomic library by hybridization to the PCR-amplified DNA product . The nucleotide sequence of the gene contained a 2,799 base-pair open reading frame that encoded a 102,274 dalton protein composed of 933 amino acids . The P . carinii ATPase protein was 69-74% identical to four fungal proton pumps but less than 35% identical to protozoan and mammalian cation transporting ATPase genes or the Ca++ ATPases of Saccharomyces . The nucleotide sequence of a portion of the TFIID gene could be translated to produce a peptide of 53 amino acids in two regions of the sequence, interrupted by a 45 bp intron . The predicted TFIID amino acid sequence was identical to yeast TFIID genes in this region. J Protozool, 1991 Nov-Dec, 38(6), 154S - 157S Inhibition of Pneumocystis dihydrofolate reductase by analogs of pyrimethamine, methotrexate and trimetrexate; Queener SF; Dihydrofolate reductase was obtained from Pneumocystis carinii isolated from heavily infected lungs of female Sprague-Dawley rats infected by transtracheal inoculation . The enzyme differed significantly from other forms of dihydrofolate reductase in response to KCl and to antifolate drugs . Dihydrofolate reductase from P . carinii was used to assess activity of analogs of pyrimethamine, methotrexate, and trimetrexate . One pyrimethamine analog was selective for P . carinii dihydrofolate reductase; potency was in the micromolar range . In contrast, 21 methotrexate analogs and 2 trimetrexate analogs were selective for P . carinii dihydrofolate reductase; potencies for these were in the nanomolar range. Trends Biochem Sci, 1991 Nov, 16(11), 430 - 5 Transcriptional regulation in the eukaryotic cell cycle; McKinney JD et al.; Cell-cycle-regulated transcription is a characteristic feature of complex cell cycles in organisms as divergent as yeasts and humans . Increasing evidence suggests that transcriptional regulation may control key events in the eukaryotic cell cycle . In this review we will address the mechanisms that may regulate transcription during the cell cycle and the roles which periodic transcription may serve in the control of cell cycle progression. Bioessays, 1991 Nov, 13(11), 613 - 8 Control of eukaryotic DNA replication at the chromosomal level; Wanka F; A hypothesis for the control of eukaryotic DNA replication at the chromosomal level is proposed . The specific regulatory problem arises from the subdivision of the genome into thousands of individually replicating units, each of which must be duplicated a single time during S-phase . The hypothesis is based on the finding of direct repeats at replication origins . Such repeats can adopt, beyond the full-length double helical structure, another configuration exposing two single-stranded loops that provide suitable templates for the initiation of DNA replication . Any further initiation at the same origin is excluded as the single strandedness is eliminated by the replication process . Restoration of the initiable loop structure is proposed to occur by DNA-protein rearrangements involved in chromosome condensation and duplication of the chromosomal protein backbone during mitosis . A possible role of the maturation promoting factor (MPF) is suggested. Biotechnol Appl Biochem, 1991 Oct, 14(2), 234 - 42 Isolation and fundamental characteristics of a phospholipase B inhibitor from autolyzed Torulaspora delbrueckii; Maruyama M et al.; Phospholipase B inhibitor was found in the autolyzate of yeast cells, Torulaspora delbrueckii . The inhibitor was purified to homogeneity by ethanol precipitation, gel filtration with Sephadex G-10, ion-exchange chromatography with DEAE-Sephacel, and gel filtration with Asahipak GS-320 . On thin-layer chromatography the purified inhibitor was detected with the Hanes-Isherwood reagent, which is used to detect phosphorus . The activity of the inhibitor was not affected by heat treatment at 100 degrees C for 1 h . Heating at 100 degrees C for 1 h in 1 M HCl and 1 M NaOH lowered activity to 76 and 80% of the original values, respectively, but heating at 110 degrees C for 24 h in 6 M HCl completely abolished activity . The inhibitor was highly soluble in water, but practically insoluble in alcohol, acetone, ether, and chloroform . The degree of inhibition of enzyme activity was not proportional to the concentration of inhibitor . The inhibitor inhibited both membrane-bound and water-soluble phospholipase B activity from T . delbrueckii at the same level; however, the inhibitor did not inhibit the activity of phospholipase A2 from snake venom (Naja naja). J Antibiot (Tokyo), 1991 Sep, 44(9), 934 - 9 Isolation of a bromo analog of rebeccamycin from Saccharothrix aerocolonigenes; Lam KS et al.; When grown in a defined medium containing 0.05% KBr, Saccharothrix aerocolonigenes ATCC 39243 produces a novel bromo analog of rebeccamycin . This new analog, designated bromorebeccamycin, has been isolated from the culture broth and purified by vacuum liquid chromatography and column chromatography . Spectroscopic data demonstrated that bromorebeccamycin has the same structure as rebeccamycin, except for the replacement of the two chlorine atoms by bromine atoms in the molecule . Bromorebeccamycin and rebeccamycin have a similar potency and activity against P388 leukemia in the murine model. J Gen Microbiol, 1991 Aug, 137 ( Pt 8), 1805 - 13 Structure-activity relationships of the nikkomycins; Decker H et al.; The structure-activity relationships of different nikkomycins were studied to evaluate the structural requirements for a potent chitin synthase inhibitor . We investigated the transport of the nikkomycins via the peptide transport system of the yeast Yarrowia lipolytica and determined the kinetic parameters for nikkomycin Z uptake {Km = 24 microM, Vmax = 2.2 nmol min-1 (mg dry wt)-1} . We demonstrated that the beta-methyl group of the N-terminal amino acid of dipeptide nikkomycins protects the molecule against peptidase activity in crude cell-extracts of different fungi . Furthermore, the relationship between inhibition constants for chitin synthase, transport of the nikkomycins via the peptide transport system, susceptibility to degradation by cellular proteases and whole-cell activity of the nikkomycins are discussed. Biopolymers, 1991 Jul, 31(8), 1009 - 16 Information concerning the mechanism of electrophoretic DNA separation provided by quantitative video-epifluorescence microscopy; Rampino NJ; Changes in conformation, length, and mobility of individual DNA molecules during agarose gel electrophoresis were measured using video micrographs obtained by epifluorescence microscopy . Globular, V-shaped, and linear conformations of DNA are found . The mobility, upon transformation from the globular to the V-shaped conformation, decreases, suggesting a collision with a gel fiber . The duration of interaction between DNA and gel fiber is proportional to the length of DNA . Hypothetically, this proportionality underlies the size separation of DNA by agarose gel electrophoresis . DNA release from the gel fiber appears to involve the movement of the arms of the V-shaped molecule around the gel fiber . Concomitant with this movement is a length reduction the degree of which is constant for DNA of various lengths in a particular buffer milieu . The luminant densitometric profiles of DNA molecules in the V conformation show maxima at the ends and apex of the V . The unequal distribution of nucleotides along the DNA chain appears to provide the driving force for the molecular movement around the gel fiber. Appl Environ Microbiol, 1991 Jul, 57(7), 1880 - 5 Plasma membrane Mg(2+)-ATPase of Pachysolen tannophilus: characterization and role in alcohol tolerance; Barbosa MF et al.; Following cell fractionation in sucrose density gradients, plasma membrane Mg(2+)-ATPase from Pachysolen tannophilus was studied . The ATPase displayed an apparent Km for ATP of 1.42 mM and was inhibited by high concentrations of Mg2+ . The inhibitory effects of ethanol, 1-propanol, 1-butanol, and benzyl alcohol on Mg(2+)-ATPase were evaluated, and the concentration of each alcohol that inhibited ATPase activity by 50% (IC50) was determined . The IC50 decreased as the chain length of the alcohol increased . Moreover, the IC50 for ATPase activity was similar to the IC50 for growth rate, suggesting an association between impaired growth and ATPase inhibition . Almost complete inhibition of ATPase activity occurred at temperatures approaching 60 degrees C, and the optimal temperature was around 44 degrees C for ATPase from both control and ethanol-treated cells . Inclusion of 50 mM MgCl2 or CaCl2 in the medium did not rescue cells from the deleterious effects of ethanol. Klin Padiatr, 1991 Jul-Aug, 203(4), 273 - 5 Seroprevalence and significance of antibodies to hepatitis C virus in pediatric patients with malignant diseases; Schneppenheim R et al.; The prevalence of antibodies to hepatitis C virus recombinant antigen c100-3 was determined in 82 pediatric patients with malignant diseases who received blood transfusions as support during their chemotherapy . By prescreening, 12 of 82 patients were repeatedly positive . No positive correlation was established neither with the number of transfused blood units nor with surrogate markers for hepatitis NANB like ALT or anti HBc . Additional testing by alternate test assays revealed discordant results in 11 of these patients . Possible explanations, like reactivity to test-immanent coexpressed antigens of Saccharomyces cerevisae are discussed. Biotechnology (N Y), 1991 Mar, 9(3), 291 - 5 Heterologous gene expression in Hansenula polymorpha: efficient secretion of glucoamylase; Gellissen G et al.; We have introduced the glucoamylase gene (GAM1) from Schwanniomyces occidentalis into the genome of the methylotrophic yeast Hansenula polymorpha to study the potential of this organism as a host for high-level expression of a heterologous gene encoding a secretory protein . Transformants of H . polymorpha containing GAM1 under control of the formate dehydrogenase (FMD) promoter are stable and efficiently secrete an active glucoamylase that is faithfully processed and modified . Yields of up to 1.4g/l of active enzyme were obtained at cell densities of 100-130 grams dry weight per liter. Eur J Clin Nutr, 1991 Feb, 45(2), 111 - 5 Plasma vitamin B6 concentrations in Nigerian adolescents; Korede O et al.; Vitamin B6 status was assessed from dietary and plasma vitamin B6 concentration using Saccharomyces uvarum as test organism, and erythrocyte alanine amino transferase activity (E-ALAT) . The subjects participating in the study were 72 males and 30 females (aged 10-18 years) who resided in a boarding institution . Mean daily dietary vitamin B6 and protein intakes were 1.56 +/- 0.42 mg and 63.0 +/- 9.6 g respectively . The corresponding mean plasma vitamin B6 concentration was 194 +/- 44.2 nmol/l . Neither age, sex nor menarche had significant effect (P less than 0.05) on plasma vitamin B6 concentration of these adolescents . Dietary vitamin B6 but not protein intake correlated with plasma vitamin B6 (r = 0.3076, P less than 0.002) . However, low dietary vitamin B6/protein ratio (less than 0.02 mg/g) was not reflected in plasma vitamin B6 concentration, but low plasma vitamin B6 concentration (120-179 nmol/l) corresponded to low E-ALAT activity after in vitro addition of pyridoxal phosphate (E-ELAT 16 per cent) . A stimulation above 25 per cent, 16-25 per cent and below 16 per cent was used as an indicator of poor, marginal and adequate vitamin B6 status, respectively . Based on these criteria 30.7 per cent, 17.8 per cent and 51.5 per cent of subjects, with corresponding mean plasma vitamin B6 of 150 +/- 28.4, 192 +/- 8.5 and 237 +/- 18.7 nmol/l are of deficient, marginal and adequate vitamin B6 status, respectively. J Antibiot (Tokyo), 1991 Feb, 44(2), 123 - 9 Isolation of new minor benanomicins; Kondo S et al.; Four minor benanomicins, dexylosylbenanomicins A and B, 2'-demethylbenanomicin A and 7-methoxybenanomicinone have been isolated from the culture filtrate of Actinomadura sp . MH193-16F4 . Their structures were confirmed by spectral analyses . Dexylosylbenanomicins A and B were derived chemically from benanomicins A and B, respectively. Antonie Van Leeuwenhoek, 1991 Feb, 59(2), 77 - 80 Septal micropores in Zygozyma and their taxonomic significance; van der Walt JP et al.; Septal micropores or plasmadesmal canals have been observed in two species of the lipomycetaceous genus Zygozyma . The presence of these canals is considered as further evidence for the connexion between the Lipomycetaceae and the Dipodascaceae . The genus Zygozyma has been emended. Gene, 1991 Jan 15, 97(2), 183 - 9 A DNA fragment containing the ADE2 gene from Schwanniomyces occidentalis can be maintained as an extrachromosomal element; Klein RD et al.; A 4.05-kb DNA fragment containing the ADE2 gene from Schwanniomyces occidentalis was cloned into the pUC19 vector . When an ade2 strain of Sc . occidentalis was transformed with this plasmid, pADE-2 was found to integrate into the host chromosome and was also present in a variety of extrachromosomal species . These extrachromosomal elements were present in multiple copies, varied in molecular mass and were composed of polymerized forms of pADE-2 . A fragment containing the ADE2 gene was used to transform a Sc . occidentalis ade2 mutant, as either a linear or circularized molecule . The linear form integrated into the host genome, whereas the circularized form was found as a stably maintained extrachromosomal element with no evidence of integration or detectable loss of the Ade+ phenotype upon subculturing of transformed yeast under nonselective conditions for 60 generations . The ratio of the number of extrachromosomal ADE2 genes to genomic ADE2 ranged from 3.8 to 6.6. Antonie Van Leeuwenhoek, 1990 Nov, 58(4), 249 - 53 Genome comparison among species of the genus Arthroascus von Arx; Smith MT et al.; Genome comparison in strains of the genus Arthroascus indicates that two species, A . javanensis (CBS 2555, Type) and A . schoenii (CBS 7223, Type), can be recognized. Antonie Van Leeuwenhoek, 1990 Nov, 58(4), 235 - 40 Phylogenetic relationships among species of the genus Issatchenkia Kudriavzev; Peterson SW et al.; The phylogenetic relatedness of Issatchenkia spp . was estimated from partial rRNA sequences in two regions of the large subunit and one region of the small subunit . I . terricola was the most divergent species of the genus, differing from other members by 18% nucleotide differences in the highly variable 25S-635 region . These data indicate Issatchenkia to be the most divergent ascomycetous yeast genus presently known. Biochim Biophys Acta, 1990 Oct 19, 1028(3), 261 - 7 Proton-linked transport systems as sensors of changes in the membrane surface potential; Cerbon J et al.; The kinetic properties of proton linked transport systems and their relation to the membrane surface potential were studied in yeast cells . (1) The negative surface potential of cells rich in anionic phospholipids was found to be 2-times higher than that of control cells; in agreement with their 2-fold increase in the anionic/zwitterionic phospholipid ratio (A/Z) . (2) At low external concentration of substrates (high-affinity systems), higher uptake activities were observed for the anions, glutamate, aspartate and phosphate; the zwitterion glycine and the cations lysine and arginine, in both phosphatidylserine and phosphatidylinositol rich cells when compared to control cells . (3) On the other hand, at high external concentration of substrates (low-affinity systems), lower uptake activities were observed for glutamate, aspartate, phosphate and glycine in the cells rich in anionic phospholipids . (4) A decrease in Km without significant alteration in Vmax was found in the high-affinity transport systems that can be explained by the increase in proton concentration at the interface caused by the enhancement in negative surface charge of the cells rich in anionic phospholipids . (5) The mechanisms of the high-affinity proton linked transport systems are compatible with a model which is necessarily ordered, protons before anions . The low-affinity transport systems, on the other hand, follow a random order of binding . The transport systems studied behave as sensors of the changes in surface potential . The reduction of the surface potential reversed the transport alterations with the following sequence: monovalent cations less than divalent cations less than cationic local anesthetics. Can J Microbiol, 1990 Sep, 36(9), 609 - 16 {The mode of action of a nonpolyenic antifungal (desertomycin) produced by a strain of Streptomyces spectabilis}; Benallaoua S et al.; A metabolite with antifungal activity, of non polyenic macrolide structure, was extracted and purified from the culture supernatant of a soil-isolated Streptomyces spectabilis strain, BT 352 . This product was found to be related to (or being) desertomycin . Six yeast and five filamentous fungus strains were used to determine minimum concentration of the metabolite that inhibits growth by 80% (IMC); it was established at 50 micrograms/mL for the fungi and at 100 micrograms/mL or more for the yeasts tested . Short-term genotoxicity tests showed no antifungal effect on the bacterial genome, and desertomycin at concentration levels of 100 micrograms/mL or more affected protein synthesis . The antifungal metabolite had no immediate inhibiting effect upon yeast respiration, even at high concentrations; however, the respiration activity of cells grown in the presence of subinhibiting doses and collected during their growth phase was reduced by as much as 40% . Saccharomyces uvarum spheroplast regeneration in a liquid medium containing desertomycin was inhibited at doses fivefold weaker than the IMC determined with intact cells . Contrary to amphotericin B, desertomycin subinhibiting doses do not modify, and if so lightly, the yeast latent phase or the spheroplast wall regeneration phase, thus indicating a fungicidal action . Moreover, following a 30-min contact with desertomycin subinhibiting and inhibiting doses, yeasts liberated potassium in large amounts, indicating that plasma membranes were affected. J Biol Chem, 1990 Aug 5, 265(22), 12864 - 8 Transport of glucose and cellobiose by Candida wickerhamii and Clavispora lusitaniae; Freer SN et al.; The cellular location of beta-1,4-glucosidase activity from, as well as the transport of glucose and cellobiose into, cells of Clavispora lusitaniae NRRL Y-5394 and Candida wickerhamii NRRL Y-2563 was investigated . The beta-glucosidase from Cl . lusitaniae appeared to be a soluble cytoplasmic enzyme . This yeast transported both glucose and cellobiose when grown in medium containing cellobiose as the sole carbon source . Glucose, but not cellobiose, uptake was observed for cells grown on glucose . The Ks and Vmax values for cellobiose transport were different when Cl . lusitaniae was cultured either aerobically (0.11 mM, 6.28 nmol.min-1.mg-1) or anaerobically (0.25 mM, 3.88 nmol-1.min-1.mg-1) . The Ks and Vmax values for glucose transport (0.23-1.10 mM and 17.2-33.9 nmol.min-1.mg-1) also differed with the various growth conditions . The beta-glucosidase from C . wickerhamii was extracytoplasmically located . This yeast transported glucose, but not cellobiose, under all growth conditions tested . The Ks for glucose uptake was 0.13-0.28 mM when C . wickerhamii was cultured on cellobiose and 0.25-0.30 mM when cultured on glucose . The Vmax values for glucose uptake were greater for cells cultured on cellobiose (35.0-37.9 nmol.min-1.mg-1) than for cells cultured on glucose (15.6-21.4 nmol.min-1.mg-1) . Cellobiose did not inhibit glucose uptake in either yeast . Glucose partially inhibited cellobiose transport in C . lusitaniae, but only if the yeast was grown aerobically . In both yeasts, sugar transport was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and 1799, but insensitive to valinomycin. Wei Sheng Wu Xue Bao, 1990 Aug, 30(4), 312 - 3 {Mutagenesis of vitamin B2 producer by using protoplasts}; Li Q et al.; Studies on preparation, regeneration and ultraviolet-mutagenesis of protoplasts of vitamin B2 producer Eremothecium aohbyii were reported . By using a complex enzyme system (0.5% snial digestase + 0.5% cellulase), a great number of protoplasts were obtained . Ultraviolet-mutagenesising positive mutation ratio is 14.29% . The HPLC analysis indicated that a lot of mutants were screened by using ultraviolet-mutagenesis mutation of protoplasts, Vitamin B2 produced by the mutant E3 increased 116.4%. Biochem Biophys Res Commun, 1990 Jul 31, 170(2), 763 - 8 Structure and partial amino acid sequence of calf thymus DNA topoisomerase II: comparison with other type II enzymes; Austin CA et al.; The partial amino acid sequence of p140 calf thymus DNA topoisomerase II was determined by analysis of cyanogen bromide peptides . Five peptides were aligned and shared extensive homology with sequences derived from cDNA clones for the human topoisomerase II isoenzyme forms . Less homology was seen with the Drosophila, yeast and bacterial type II enzymes . Calf and human enzymes shared epitopes allowing isolation of a cDNA clone to human topoisomerase II isoenzyme alpha . Our results indicate that calf thymus p140 topoisomerase II is an active N-terminal proteolytic fragment of the native p180 enzyme and demonstrate that mammalian type II enzymes exhibit close sequence similarity. FEBS Lett, 1990 Jul 2, 267(1), 114 - 6 Essential role of ferrous iron in cyanide-resistant respiration in Hansenula anomala; Minagawa N et al.; Antimycin A-dependent induction of cyanide-resistant respiration in Hansenula anomala was completely blocked by o-phenanthroline, alpha,alpha'-dipyridyl, or 8-hydroxyquinoline . Pulse-labeling of the cells with {35S}methionine in the presence of both antimycin A and o-phenanthroline indicated that the 36-kDa protein previously reported to be involved in cyanide-resistant respiration {(1989) J . Biochem . 105, 864-866} was formed in mitochondria even under these conditions . The addition of Fe2+, but not Fe3+, ions to these cells in the presence of cycloheximide resulted in the rapid expression of cyanide-resistant respiration activity . These results suggest that in the presence of both antimycin A and o-phenanthroline an inactive form of the 36-kDa protein was formed and Fe2+ ions converted it to the active form . It is also likely that Fe2+ ions are involved in the reaction mechanism of cyanide-resistant respiration. Yeast, 1990 Jul-Aug, 6(4), 299 - 310 Dekkera, Brettanomyces and Eeniella: electrophoretic comparison of enzymes and DNA-DNA homology; Smith MT et al.; The taxonomic status of various species of Dekkera, Brettanomyces and Eeniella was examined by electrophoretic comparison of enzymes, by deoxyribonucleic acid homology and by physiological characterization . These studies demonstrated that two teleomorphic Dekkera species, D . anomala and D . bruxellensis (synonym D . intermedia), and four anamorphic Brettanomyces species, B . anomalus (synonym B . claussenii), B . bruxellensis (synonym B . abstinens, B . custersii, B . intermedius, B . lambicus), B . custersianus and B . naardenensis, can be recognized . The anamorphic genus Eeniella remained as a separate, monotypic taxon. J Mol Evol, 1990 Jun, 30(6), 522 - 62 Evolution of EF-hand calcium-modulated proteins . I . Relationships based on amino acid sequences; Moncrief ND et al.; The relationships among 153 EF-hand (calcium-modulated) proteins of known amino acid sequence were determined using the method of maximum parsimony . These proteins can be ordered into 12 distinct subfamilies--calmodulin, troponin C, essential light chain of myosin, regulatory light chain, sarcoplasmic calcium binding protein, calpain, aequorin, Stronglyocentrotus purpuratus ectodermal protein, calbindin 28 kd, parvalbumin, alpha-actinin, and S100/intestinal calcium-binding protein . Eight individual proteins--calcineurin B from Bos, troponin C from Astacus, calcium vector protein from Branchiostoma, caltractin from Chlamydomonas, cell-division-cycle 31 gene product from Saccharomyces, 10-kd calcium-binding protein from Tetrahymena, LPS1 eight-domain protein from Lytechinus, and calcium-binding protein from Streptomyces--are tentatively identified as unique; that is, each may be the sole representative of another subfamily . We present dendrograms showing the relationships among the subfamilies and uniques as well as dendrograms showing relationships within each subfamily . The EF-hand proteins have been characterized from a broad range of organismal sources, and they have an enormous range of function . This is reflected in the complexity of the dendrograms . At this time we urge caution in assigning a simple scheme of gene duplications to account for the evolution of the 600 EF-hand domains of known sequence. FEBS Lett, 1990 May 7, 264(1), 149 - 52 A 36-kDa mitochondrial protein is responsible for cyanide-resistant respiration in Hansenula anomala; Minagawa N et al.; Antimycin A-dependent induction of cyanide-resistant respiration in Hansenula anomala was reversibly blocked by carbonylcyanide-m-chlorophenylhydrazone (CCCP) . When the cells were pulse-labeled with {35S}methionine in the presence of both antimycin A and CCCP, the radioactivity was incorporated into a 39 kDa mitochondrial protein . Upon removal of CCCP, this protein was processed into a 36 kDa form . The increase in the 36 kDa protein completely paralleled that in cyanide-resistant respiration activity, suggesting that the 39 kDa protein is the precursor of the 36 kDa protein, which is responsible for cyanide-resistant respiration. Biotechnol Prog, 1990 May-Jun, 6(3), 205 - 9 Experimental and theoretical evidence for convective nutrient transport in an immobilized cell support; Bringi V et al.; Even though immobilized-cell reactors possess several engineering advantages over free-cell reactors, their full potential has not been realized because mass transfer often limits the rate of nutrient supply and product removal from immobilized cell supports . We studied the interaction between mass transfer and reaction kinetics in the anaerobic conversion of glucose to CO2 and ethanol by yeast immobilized in a porous rotating disk on the agitator shaft of a conventional CSTR . A Sherwood number correlation was used to show that external mass-transfer resistances were negligible under typical operating conditions . The modulus of Weisz based on observable reaction parameters was used to gauge the importance of pore diffusion limitations . Under conditions for which significant pore diffusion effects and hence low effectiveness factors (eta = ca . 0.1) would be predicted, the observed reaction rates were much higher than expected (eta = ca . 1), suggesting that pore diffusion limitations were at least partially relieved by convective transport of glucose into the support . Two possible mechanisms of convective transport are discussed . We hypothesize that gas evolution was responsible for the convective enhancement of glucose supply. Antonie Van Leeuwenhoek, 1990 Apr, 57(3), 131 - 7 Alternative respiration pathways in Schwanniomyces castellii . II . Characteristics of oxidation pathways; Dubreucq E et al.; By using cytochrome-deficient mutants of Schwanniomyces castellii found previously, we measured the inhibition constants of azide and SHAM-alone or combined-for the different oxidative pathways, in order to determine the more suitable concentrations of inhibitors . This allowed us to measure the real capacity of each pathway . We calculated their affinity for oxygen, and determined that O2 was preferencially reduced by the cytochromic pathway, then by the SHAM-sensitive pathway, and finally by the SHAM+AA-insensitive pathway. Antonie Van Leeuwenhoek, 1990 Apr, 57(3), 123 - 30 Alternative respiration pathways in Schwanniomyces castellii . I . Isolation and characterization of cytochrome-deficient mutants; Dubreucq E et al.; We have isolated and studied cytochromic-deficient mutants of the amylolytic yeast Schwanniomyces castellii in order to study the possible contribution of cytochromes to alternative pathways . Three mutants were found, lacking cytochrome b, a + a3, or b and a + a3 . All strains presented two alternative pathways, which were induced in the wild strain when cytochromic respiration was suppressed by growth in the presence of inhibitors, or without copper . If cytochromic respiration was absent, the Yxs yields in aerobiosis were higher than in anaerobiosis . This shows that the alternative pathways play a part in energy conservation . Cytochrome a + a3 did not appear to be directly involved in the alternative pathways. Biochim Biophys Acta, 1990 Mar 29, 1038(1), 74 - 9 Electron microscopy and image analysis of two-dimensional crystals and single molecules of alcohol oxidase from Hansenula polymorpha; Vonck J et al.; The octameric protein alcohol oxidase from the yeast Hansenula polymorpha was studied by electron microscopy and image analysis . Two-dimensional crystals were formed by applying the protein, in a phosphate buffer containing poly(ethylene glycol) and EDTA, to a carbon-coated formvar film which had been glow-discharged in pentylamine at least several hours earlier . The crystals show p4 symmetry and have a unit cell of 12.5 X 12.5 nm2, containing one molecule . Image analysis of the crystals and of single molecules yielded two different views . From these it can be deduced that the subunits have an elongated shape and form two layers of four, stacked face to face . A tentative model of the structure is presented. Genetika, 1990 Mar, 26(3), 424 - 32 {Genetic strains of Hansenula polymorpha}; Bodunova EN et al.; Six centromeric linkage groups and four non-centromeric fragments are revealed in the genetic stocks of Hansenula polymorpha which were obtained by intratetrad breeding in several generations of two genetically different parental strains progeny . Fourteen nuclear markers are mapped, including auxotrophic mutations, mating regulation loci, determinants of sporulation and heat tolerance . Complex origin of the haploid genome of these stocks leads to affinity interactions and to 14 per cent increase in DNA content in haploid stocks, as compared with the parental strains. Cell, 1990 Jan 26, 60(2), 319 - 28 Snap helix with knob and hole: essential repeats in S . pombe nuclear protein nuc2+; Hirano T et al.; The S . pombe nuc2+ gene is required for mitotic chromosome disjunction . Its mutation arrests mitosis at the metaphase . The gene product is present in the nuclear scaffold-like fraction . The nuc2+ protein contains a domain, separated from ten 34 amino acid repeat segments, that is capable of binding AT-rich DNA in vitro . The ts mutation resides in one of the 34 amino acid repeats . Circular dichroism, limited proteolysis of the repeats, and model fitting indicate the presence of helical segments connected by protease-sensitive hinges . We propose that these repeats form a novel secondary structure (snap helix) having "knob and hole" helix-associating motifs . The packing of the snap helices would be stabilized by the bonding between the hydrophobic amino acids surrounding the knobs and holes . The nuc2+ protein may in one way bind to DNA and in another way mutually associate to form a part of the chromosome scaffold. Ukr Biokhim Zh, 1990 Jan-Feb, 62(1), 108 - 12 {The effect of citrate and dihydroxyacetone on methanol oxidation in Hansenula polymorpha}; Sibirnyi AA et al.; Citrate and dihydroxyacetone inhibited {14C} incorporation from radioactive methanol to CO2 by washed cells of methylotrophic yeast Hansenula polymorpha grown in the media containing mixture of methanol with citrate or dihydroxyacetone, respectively . These results are discussed in connection with the earlier hypothesis on participation of the tricarboxylic acid cycle in the energy supply of the methylotrophic growth. J Biol Chem, 1989 Nov 15, 264(32), 19366 - 72 Structure and expression of fungal calmodulin gene; LeJohn HB; I report on the isolation, structural analysis, and in vivo expression patterns of a fungal calmodulin gene . The gene is intronless and encodes a protein of 148 amino acid residues that is 92% homologous with vertebrate calmodulins . Through S1 nuclease transcript mapping, it was determined that the cloned gene (a) is transcribed in vivo, (b) has a 5'-untranslated region of about 400 nucleotides, and (c) has a 3'-untranslated end of about 300 nucleotides . Southern blot hybridization analysis of the genomic DNA and the cloned gene provide evidence for the existence of only one type of calmodulin gene in the organism . The amino acid sequence deduced from the DNA sequence shows that Achlya klebsiana calmodulin has amino acid substitutions that are a mix of those seen in calmodulins from invertebrates such as Drosophila and trypanosome when compared to mammalian calmodulins . Not surprisingly, it has less resemblance to calmodulins from Saccharomyces and Dictyostelium. J Biol Chem, 1989 Nov 15, 264(32), 19407 - 15 Intact DNA polymerase alpha/primase from mouse cells . Purification and structure; Goulian M et al.; A procedure is described for purification of DNA polymerase alpha/primase from cultured mouse lymphoblasts . Approximately 0.5 mg of enzyme, free of detectable contaminants, was obtained from 40 g of cells using seven conventional purification steps . The mouse enzyme contains subunits of 180, 70, 56, and 47 kDa, almost completely intact and similar to the subunit sizes reported for DNA polymerase alpha/primase from Drosophila embryos (Kaguni, L . S., Rossignol, J-M., Conaway, R . C., and Lehman, I . R . (1983) Proc . Natl . Acad . Sci . U.S.A . 80, 2221-2225) and Saccharomyces (Plevani, P., Foiani, M., Valsasnini, P., Badaracco, G., Cheriathundam, E., and Chang, L.M.S . (1985) J . Biol . Chem . 260, 7102-7107) . A similar structure has also been inferred for mammalian DNA polymerase alpha/primase; however, previous descriptions of the highly purified mammalian enzyme complex have included substantial evidence of proteolysis and/or partial loss of subunits . In particular, the intact 180-kDa subunit has ordinarily been a minor component, and the molecular mass of the complex and total number of subunits have not been established . Results reported here indicate that the native DNA polymerase alpha/primase consists of one each of the four subunit sizes for a total of 353 kDa, based on estimates from denaturing gels, or 344 kDa when sizes deduced from available nucleic acid sequence data are substituted for three of the four subunits . A figure of 313 kDa was calculated from the sedimentation coefficient (8.9 S) and Stokes radius (81.1 A), the values for which also indicate a frictional ratio of 1.80, corresponding to an axial ratio of approximately 16 and suggesting a highly extended structure. Dtsch Tierarztl Wochenschr, 1989 Sep, 96(8), 416 - 9 {In vitro parameters for the comprehension of the immunocompetence of chickens of different lines . 2 . Testing an immunocompetent parameter as a selection criterion in the chicken}; Kahl D et al.; A cytotoxicity test using blood and saccharomyces cells (SCT) was experimentally applied in a sample of pedigreed commercial chickens from different lines over 3 generations to test its usefulness as a parameter for the nonspecific defense capacity . The results clearly show a significant difference between the SC rates in the different lines . Positive heritabilities exist between the SC rates of the 3 generations . The correlations between SC rates of males in the first generation and parameters of layer productivity of their daughters are nonsignificant . The possibility to integrate the parameter SC into the selection index is critically discussed. J Biochem (Tokyo), 1989 Aug, 106(2), 197 - 204 Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat mitochondrial acetoacetyl-CoA thiolase; Fukao T et al.; cDNA clones for rat mitochondrial acetoacetyl-CoA thiolase were isolated and sequenced . The most 5'-extended clone (RT2-6) consisted of 1,460 bases and contained a 1,272-base open reading frame encoding a polypeptide of 424 amino acid residues . A coupled in vitro transcription/translation analysis of RT2-6 revealed that RT2-6 encodes the entire precursor of this enzyme . The amino-terminal sequence and amino acid composition of the purified enzyme agreed with the primary structure deduced from the cDNA . The calculated molecular masses of the precursor and the subunit of the mature enzyme are 44,694 and 41,364 Da, respectively . The primary structure of this enzyme was compared with those of four other thiolases (rat mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases, acetoacetyl-CoA thiolase of Zoogloea ramigera, and cytosolic acetoacetyl-CoA thiolase of Saccharomyces uvarum) . Marked homology between any two of them (34-51% identity) indicates that the genes of thiolases have evolved from a common ancestral gene . It has been reported that this enzyme has two isoenzymes A and B . However, the purified isoenzymes were indistinguishable from each other in some analyses . Though 17 independent cDNA clones were isolated, no definite evidence indicating the presence of different cDNAs was found. Genetics, 1989 Aug, 122(4), 773 - 82 cdc2 and the regulation of mitosis: six interacting mcs genes; Molz L et al.; A cdc2-3w weel-50 double mutant of fission yeast displays a temperature-sensitive lethal phenotype that is associated with gross abnormalities of chromosome segregation and has been termed mitotic catastrophe . In order to identify new genetic elements that might interact with the cdc2 protein kinase in the regulation of mitosis, we have isolated revertants of the lethal double mutant . The suppressor mutations define six mcs genes (mcs: mitotic catastrophe suppressor) that are not allelic to any of the following mitotic control genes: cdc2, wee 1, cdc13, cdc25, suc1 or nim1 . Each mcs mutation is recessive with respect to wild-type in its ability to suppress mitotic catastrophe . None confer a lethal phenotype as a single mutant but few of the mutants are expected to be nulls . A diverse range of genetic interactions between the mcs mutants and other mitotic regulators were uncovered, including the following examples . First, mcs2 cdc2w or mcs6 cdc2w double mutants display a cell cycle defect dependent on the specific wee allele of cdc2 . Second, both mcs1 cdc25-22 or mcs4 cdc25-22 double mutants are nonconditionally lethal, even at a temperature normally permissive for cdc25-22 . Finally, the characteristic suppression of the cdc25 phenotype by a loss-of-function wee1 mutation is reversed in a mcs3 mutant background . The mcs genes define new mitotic elements that might be activators or substrates of the cdc2 protein kinase. Biochim Biophys Acta, 1989 Jul 7, 1008(2), 157 - 67 Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha; Bruinenberg PG et al.; We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha . The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H . polymorpha genomic library . The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined . The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435 . The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides . The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein . Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies. Cell, 1989 Jun 2, 57(5), 739 - 51 Composite motifs and repeat symmetry in S . pombe centromeres: direct analysis by integration of NotI restriction sites; Chikashige Y et al.; S . pombe centromeres are large and complex . We introduced a method that enables us to characterize directly centromere DNAs . Genomic DNA fragments containing cen1, cen2, or cen3, respectively, are made by cleaving NotI sites integrated on target sites and are partially restricted for long-range mapping in PFG electrophoresis . The 40 kb long cen1 consists of two inverted approximately 10 kb motifs, each containing centromeric elements dg and dh, flanked by a central region . In cen2, three motifs are arranged in inverted and direct orientations with flanking domains, making up the approximately 70 kb long repetitious region . In cen3, approximately 15 copies of dg-dh constitute a region longer than 100 kb . A set of inverted motifs with an approximately 15 kb central region might be a prototype for the S . pombe centromeres . The motifs appear to play a role in chromosome stability and segregation . Their action may be additive, and the mutual directions of dg and dh inside a motif may not be essential for function. Eur J Epidemiol, 1989 Jun, 5(2), 239 - 43 Fungal air-borne spores as health risk factors among workers in alimentary industries; Palmas F et al.; A survey to evaluate the occurrence of air-borne fungal spores in two different food industries, dairies and bakeries, was conducted . Our data revealed considerable fungal pollution in the environments of both industries, as well as some differences in the distribution of the genera of fungi recovered . Noteworthy was the frequent finding of numerous fungi frequently responsible for allergic rhinitis, asthma and other diseases, or well-known for their production of mycotoxins in foods or characterized by their degradative activity against various substances . Aspergillus, Candida, Fusarium, Geotrichum, Mucor and Penicillium were the most common genera identified in dairies while Alternaria, Aspergillus, Botrytis, Candida, Cladosporium, Penicillium and Saccharomyces occurred more frequently in bakeries . The survey showed that fungi can play a significant role in allergic and non-allergic diseases in modern working environments. J Nutr Sci Vitaminol (Tokyo), 1989 Jun, 35(3), 171 - 80 Determination of vitamin B6 derivatives in foods and biological materials by reversed-phase HPLC; Toukairin-Oda T et al.; Through use of a simplified analyzing system, seven vitamin B6 derivatives were determined with a satisfactory sensitivity and precision . This system consisted of a single reversed-phase ODS column with a fluorescence detector employing an isocratic solvent system . Each vitamin B6 derivative in some foods and biological materials was determined, based on the measurement of the integrated peak area . The data obtained by this method were compared with those obtained from a bioassay by Saccharomyces uvarum ATCC 9080, after acid hydrolysis of these materials. Eur J Biochem, 1989 Jun 1, 182(1), 67 - 75 Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b2 after mild proteolysis by an H . anomala proteinase; Celerier J et al.; The protomeric chain of Hansenula anomala flavocytochrome b2 was previously shown to be built as the covalent association of two functional domains: an L-lactate dehydrogenase domain and a cytochrome c reductase domain, joined together by a proteolytically sensitive zone . This paper concerns the specific cleavage of this latter zone with a H . anomala proteinase(s) preparation and the purification of the resulting L-lactate dehydrogenase moiety of the molecule with at least 25% recovery, (i.e . one order of magnitude more than for the previously published method) . A preliminary characterization of this dehydrogenase domain indicates that it is a tetramer (Mr = 4 x 39000) containing FMN as expected and not heme . It has high L-lactate:ferricyanide oxidoreductase activity (about 70% that of the whole flavocytochrome b2) and the same Km for L(+)-lactate as flavocytochrome b2, but it has no L-lactate:cytochrome c oxidoreductase activity . Its flavin semiquinone is stabilized in the presence of pyruvate as in flavocytochrome b2 . The subcellular origin of the H . anomala proteinase in the preparation has not yet been elucidated. Eur J Biochem, 1989 Jun 1, 182(1), 57 - 65 The effects of multiple amino acid substitutions on the polypeptide backbone of tuna and horse cytochromes c; Gao Y et al.; The cytochromes c provide a wide range of natural and mutant homologous proteins which may be used to study structure/function relationships in biological electron-transfer reactions . A description of the cytochrome c structure has been provided by high-resolution X-ray crystallography for the cytochromes from tuna, bonito, rice and yeast (Saccharomyces iso-1) . Correlation of these structures with NMR parameters is necessary to confirm the structure of the protein in solution and to permit the routine characterisation of cytochromes c with novel sequences . We have previously reported a method based on the analysis of pseudocontact shifts which allowed us to compare the conformations of some amino acid side chains of tuna cytochrome c in solution and in the crystalline state . Here we report a comparison of the conformations of the polypeptide backbone of cytochromes c in proteins from tuna and horse, using the chemical shifts of the amide NH and C alpha H protons . It is found that the backbone conformation and hydrogen-bond network is closely conserved between these proteins, despite 19 amino acid substitutions . Appreciable differences occur in two regions, around Asn 31 and at the beginning of the 60s helix . Evidence for some rotational or translational motion of the C-terminal helix is also presented. Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1989 May, 22(2), 116 - 31 Macromolecule synthesis in temperature-sensitive mutants of methanol-utilizing Hansenula polymorpha; Yang SS et al.; Temperature sensitive (ts) mutants of methanol-utilizing Hansenula polymorpha NTU-AM-P5 were isolated by UV irradiation, EMS and/or NTG treatment . They grow normally at permissive temperature (PT) 38 degrees C, but can not grow at restrictive temperature (RT) 46 degrees C . From chemical composition analysis, it was found that ts mutants NTU-AM-L2 and NTU-AM-L3 had higher RNA content than the others; while ts mutant NTU-AM-E19 had the highest protein content among the isolated strains . Leucinyl-tRNA synthetases activity was the highest among the twenty aminoacyl-tRNA synthetases in both wild type and their ts mutants . When the cells were shifted from PT to RT for 12 h, total aminoacyl-tRNA synthetase activity decreased in all tested strains . Leucinyl-tRNA synthetase of ts mutant NTU-AM-E10 decreased 91.23% . At RT, it was found that ts mutants NTU-AM-E10 and NTU-AM-E20 were defective in DNA synthesis; ts mutants NTU-AM-E15, NTU-AM-E20, NTU-AM-N37 and NTU-AM-m5 were defective in RNA synthesis; ts mutants NTU-AM-E10, NTU-AM-E20 and NTU-AM-m5 were somewhat defective in protein synthesis; while ts mutants NTU-AM-L2 and NTU-AM-L3 did not belong to any one of the above classifications. Electrophoresis, 1989 May-Jun, 10(5-6), 302 - 9 Pulsed field gel electrophoresis: studies of DNA migration made with the programmable, autonomously-controlled electrode electrophoresis system; Birren BW et al.; We have studied the migration of DNA in pulsed field agarose gels under a variety of electrophoresis conditions . We have made use of an instrument which can generate electric fields of any orientation, magnitude, or duration to compare different separation techniques for DNA molecules of from 1 to several thousand kilobase pairs . We discuss the capabilities of the system and present results of gel runs in which electrophoresis conditions were changed individually or in combination . The mobility of DNA in pulsed field gels is shown to reflect a number of interdependent physical parameters. Cell, 1989 Apr 7, 57(1), 177 - 87 Genetic control of cell division patterns in the Drosophila embryo; Edgar BA et al.; In Drosophila embryogenesis, mitotic control undergoes a significant transition during the 14th interphase . Mitoses before interphase 14 run on maternal products, and occur in metasynchronous waves . Mitoses after interphase 14 require zygotic transcription, and occur asyncronously in an intricate, highly ordered spatio-temporal pattern . Mutations at the string (stg) locus cause cell-cycle arrest during this transition, in G2 of interphase 14, yet do not arrest other aspects of development . This phenotype suggests that stg is required specifically for initiating mitosis . We describe the cloning of stg, and show that its predicted amino acid sequence is homologous to that of cdc25, a regular of mitotic initiation in the yeast S . pombe . In addition, we show that zygotic expression of stg mRNA occurs in a dynamic series of spatial patterns which anticipate the patterns of the zygotically driven cell divisions . Therefore we suggest that regulated expression of stg mRNA controls the timing and location of these embryonic cell divisions. Eur J Biochem, 1989 Apr 1, 180(3), 535 - 45 Domain structure of mitochondrial and chloroplast targeting peptides; von Heijne G et al.; Representative samples of mitochondrial and chloroplast targeting peptides have been analyzed in terms of amino acid composition, positional amino acid preferences and amphiphilic character . No highly conserved 'homology blocks' are found in either class of topogenic sequence . Mitochondrial-matrix-targeting peptides are composed of two domains with different amphiphilic properties . Arginine is frequently found either at position -10 or -2 relative to the cleavage site, suggesting that some targeting peptides may be cleaved twice in succession by two different matrix proteases . In stroma-targeting chloroplast transit peptides three distinct regions are evident: an uncharged amino-terminal domain, a central domain lacking acidic residues and a carboxy-terminal domain with the potential to form an amphiphilic beta-strand . Targeting peptides that route proteins to the mitochondrial intermembrane space or the lumen of chloroplast thylakoids have a mosaic design with an amino-terminal matrix- or stroma-targeting part attached to a carboxy-terminal extension that shares many characteristics with secretory signal peptides. Antonie Van Leeuwenhoek, 1989 Apr, 55(4), 369 - 82 Species delineation in the genus Nadsonia Sydow; Golubev WI et al.; The genus Nadsonia Sydow is revised on the basis of morphology, physiology, amino acid and fatty acid composition, electrophoretic patterns of some enzymes and DNA relatedness . Two species, N . commutata (type CBS 6640) and N . fulvescens, with two varieties, N . fulvescens var . fulvescens (type CBS 2596) and N . fulvescens var . elongata (type CBS 2594) nov . comb . are recognized . A modified diagnosis of the genus and a key are given. FEBS Lett, 1989 Feb 13, 244(1), 213 - 6 Use of electron microscopy in the examination of lattice defects in crystals of alcohol oxidase; Van der Klei IJ et al.; Alcohol oxidase, purified from the yeast Hansenula polymorpha, was crystallized in vitro for the purpose of determining its structure at atomic resolution by X-ray diffraction methods . The crystals obtained yielded only extremely weak diffraction patterns: the maximal resolution observed was in the best case 6 A . Electron microscopy of thin sections indicated that most crystals showed lattice defects which might explain the poor diffraction patterns: most surprising was the appearance of large holes interrupting an otherwise regular lattice in one of the crystal forms examined . Our results indicate that transmission electron microscopy is a suitable tool for the inspection of crystals to be used in X-ray crystallography . The method allows rapid determination of lattice defects and enables optimization of crystallization conditions. J Bacteriol, 1989 Feb, 171(2), 1173 - 7 Purification and properties of glutathione transferase from Issatchenkia orientalis; Tamaki H et al.; Glutathione transferase (GST) (EC 2.5.1.18) was purified from a cell extract of Issatchenkia orientalis, and two GST isoenzymes were isolated . They had molecular weights of 37,500 and 40,000 and were designated GST Y-1 and GST Y-2, respectively . GST Y-1 and GST Y-2 gave single bands with molecular weights of 22,000 and 23,500, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . GST Y-1 and GST Y-2 were immunologically distinguished from each other . GST Y-1 showed specific activity 10.4-times and 6.0-times higher when 1-chloro-2,4-dinitrobenzene and o-dinitrobenzene were used as substrates, respectively, than GST Y-2 . GST acti