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Tian Li, Ying-Ying Guo, Guan-Qing Qiao, Guo-Qiang Chen
5-Aminolevulinic acid (ALA), an important cell metabolic intermediate useful for cancer treatments or plant growth regulator, was produced by recombinant Escherichia coli expressing the codon optimized mitochondrial 5-aminolevulinic acid synthase (EC:, hem1) from Saccharomyces cerevisiae controlled via the plasmid encoding T7 expression system with a T7 RNA polymerase. When a more efficient autoinduced expression approach free of IPTG was applied, the recombinant containing antibiotic-free stabilized plasmid was able to produce 3...
November 18, 2016: ACS Synthetic Biology
Shu Qin Toh, Geoffrey N Gobert, David Malagón Martínez, Malcolm K Jones
Schistosomes ingest host erythrocytes, liberating large quantities of haem. Despite its toxicity, haem is an essential factor for numerous biological reactions, and may be an important iron source for these helminths. We used a fluorescence haem analogue, palladium mesoporphyrin, to investigate pathways of haem acquisition, and showed that palladium mesoporphyrin accumulates in the vitellaria (eggshell precursor glands) and ovary of female Schistosoma mansoni. Furthermore, incubation of adult females in 10-100 μm cyclosporin A (IC50 = 2...
September 2015: FEBS Journal
Magdalena Marek, Daniele Silvestro, Maria D Fredslund, Tonni G Andersen, Thomas G Pomorski
Sterol uptake in fungi is a multistep process that involves interaction between external sterols and the cell wall, incorporation of sterol molecules into the plasma membrane, and subsequent integration into intracellular membranes for turnover. ATP-binding cassette (ABC) transporters have been implicated in sterol uptake, but key features of their activity remain to be elucidated. Here, we apply fluorescent cholesterol (NBD-cholesterol) to monitor sterol uptake under anaerobic and aerobic conditions in two fungal species, Candida glabrata (Cg) and Saccharomyces cerevisiae (Sc)...
December 2014: FEMS Yeast Research
D Urban-Grimal, V Ribes, R Labbe-Bois
We have cloned the structural gene HEM1 for 5-aminolevulinate (ALA) synthase from Saccharomyces cerevisiae by transformation and complementation of a yeast hem1-5 mutant which was previously shown to lack ALA synthase activity (Urban-Grimal and Labbe Bois 1981) and had no immunodetectable ALA synthase protein when tested with yeast ALA synthase antiserum. The gene was selected from a recombinant cosmid pool which contained wild-type yeast genomic DNA fragments of an average size of 40 kb. The cloned gene was identified by the restauration...
July 1984: Current Genetics
R Gudenus, A Spence, A Hartig, M Smith, H Ruis
A Saccharomyces cerevisiae mutant (hem1 cycl-1) was transformed with plasmids bearing a chromosomal centromer (CEN3) and a 2 μm DNA replication origin. In one of the plasmids a functional CYC1 gene was present, in a second plasmid an XhoI fragment located between bases -245 and -678 upstream from the translation initiation codon had been deleted, in a third plasmid this region had been inverted. Results of hybridization experiments carried out with mRNA isolated from heme-deficient and heme-containing transformants indicated that heme controls transcription of the CYC1 gene and that DNA sequences located within the upstream XhoI fragment are involved in activation of the gene by heme...
January 1984: Current Genetics
M Arrese, E Carvajal, S Robison, A Sambunaris, A Panek, J Mattoon
HEM1, the structural gene for δ-aminolevulinic acid synthase, has been isolated on recombinant plasmids. A yeast genomic pool constructed in the E. coli - yeast shuttle vector YEp13 was used to clone the HEM1 gene by complementation. A leu2 hem1 yeast mutants was transformed with the yeast genomic pool and hybrid YEp13 plasmids carrying the HEM1 gene were cloned by their ability to complement both the leu2 and hem1 mutations in the recipient strain. The yeast transformants, bearing the HEM1-containing plasmids pYe(HEM1), showed a 24-28 fold increase in δ-aminolevulinic acid synthase activity and in the intracellular content of δ-aminolevulinic acid (5-8 fold) as compared to wild type strains, suggesting that the p(HEM1) gene is being expressed as a catalytically active enzyme which can be transported into the mitochondria...
June 1983: Current Genetics
Maia M Chan, Jason M Wooden, Mark Tsang, Diana M Gilligan, Dinesh K Hirenallur-S, Greg L Finney, Eric Rynes, Michael Maccoss, Julita A Ramirez, Heon Park, Brian M Iritani
Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan...
2013: PloS One
Miroslava Spanova, Dagmar Zweytick, Karl Lohner, Lisa Klug, Erich Leitner, Albin Hermetter, Günther Daum
In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127-6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes...
April 2012: Biochimica et Biophysica Acta
Peter Kohut, Daniel Wüstner, Lucia Hronska, Karl Kuchler, Ivan Hapala, Martin Valachovic
Uptake of external sterols in the yeast Saccharomyces cerevisiae is a multistep process limited to anaerobiosis or heme deficiency. It includes crossing the cell wall, insertion of sterol molecules into plasma membrane and their internalization and integration into intracellular membranes. We applied the fluorescent ergosterol analog dehydroergosterol (DHE) to monitor the initial steps of sterol uptake by three independent approaches: fluorescence spectroscopy, fluorescence microscopy and sterol quantification by HPLC...
January 7, 2011: Biochemical and Biophysical Research Communications
Zhi-Ping Zhang, Quan-Hong Yao, Liang-Ju Wang
A gene encoding aminolevulinate synthase (ALA-S) in yeast (Saccharomyces cerevisiae YHem1) was introduced into the genome of tobacco (Nicoliana tabacum) under the control of Arabidopsis thaliana HemA1 gene promoter (AtHemA1 P). All transgenic lines transcribed the YHem1 gene, especially under light condition. The capacity to synthesize ALA and therefore chlorophyll was increased in transformed plants. Determination of gas exchange suggested that transgenic plants had significantly higher level of net photosynthetic rate (P ( n )), stomatal conductance (G ( s )) and transpiration rate (T ( r )), compared to the wild type (WT)...
October 2011: Molecular Biology Reports
Zhi-Ping Zhang, Quan-Hong Yao, Liang-Ju Wang
5-Aminolevulinate (ALA) is well-known as an essential biosynthetic precursor of all tetrapyrrole compounds, which has been suggested to improve plant salt tolerance by exogenous application. In this work, the gene encoding aminolevulinate synthase (ALA-S) in yeast (Saccharomyces cerevisiae Hem1) was introduced into the genome of Arabidopsis controlled by the Arabidopsis thaliana HemA1 gene promoter. All transgenic lines were able to transcribe the YHem1 gene, especially under light condition. The chimeric protein (YHem1-EGFP) was found co-localizing with the mitochondria in onion epidermal cells...
May 2010: BMB Reports
Liang-da Chiu, Hiro-o Hamaguchi
HEM1 gene encodes δ-aminolevulinate synthase that is required for haem synthesis. It is an essential gene for yeast survival. The Raman spectra of HEM1 knockout (hem1Δ) yeast cells lacks a Raman band at 1602 cm(-1) that has been shown to reflect cell metabolic activity. This result suggests that the molecule giving rise to the"Raman spectroscopic signature of life" is closely related to haem functions in the cell. High amount of squalene is also observed in the hem1Δ strain, which is another new discovery of this study...
January 2011: Journal of Biophotonics
Heon Park, Karen Staehling-Hampton, Mark W Appleby, Mary E Brunkow, Tania Habib, Yi Zhang, Fred Ramsdell, H Denny Liggitt, Brian Freie, Mark Tsang, George Carlson, Sherree Friend, Charles Frevert, Brian M Iritani
Hem1 (Hematopoietic protein 1) is a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins. Orthologues of Hem1 in Dictyostelium discoideum, Drosophila melanogaster, and Caenorhabditis elegans are essential for cytoskeletal reorganization, embryonic cell migration, and morphogenesis. However, the in vivo functions of mammalian Hem1 are not known. Using a chemical mutagenesis strategy in mice to identify novel genes involved in immune cell functions, we positionally cloned a nonsense mutation in the Hem1 gene...
November 24, 2008: Journal of Experimental Medicine
Avadhut D Joshi, Ganapati V Hegde, John D Dickinson, Amit K Mittal, James C Lynch, James D Eudy, James O Armitage, Philip J Bierman, R Gregory Bociek, Marcel P Devetten, Julie M Vose, Shantaram S Joshi
PURPOSE: In B-cell chronic lymphocytic leukemia (CLL), high CD38 expression has been associated with unfavorable clinical course, advanced disease, resistance to therapy, shorter time to first treatment, and shorter survival. However, the genes associated with CLL patient subgroups with high and low CD38 expression and their potential role in disease progression is not known. EXPERIMENTAL DESIGN: To identify the genes associated with the clinical disparity in CLL patients with high versus low CD38 expression, transcriptional profiles were obtained from CLL cells from 39 different patients using oligonucleotide microarray...
September 15, 2007: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
Orna Ben-Naim, Ravit Eshed, Anna Parnis, Paula Teper-Bamnolker, Akiva Shalit, George Coupland, Alon Samach, Eliezer Lifschitz
CONSTANS-Like (COL) proteins are plant-specific nuclear regulators of gene expression but do not contain a known DNA-binding motif. We tested whether a common DNA-binding protein can deliver these proteins to specific cis-acting elements. We screened for proteins that interact with two members of a subgroup of COL proteins. These COL proteins were Tomato COL1 (TCOL1), which does not seem to be involved in the control of flowering time, and the Arabidopsis thaliana CONSTANS (AtCO) protein which mediates photoperiodic induction of flowering...
May 2006: Plant Journal: for Cell and Molecular Biology
Sonja Reiner, Delphine Micolod, Günther Zellnig, Roger Schneiter
The mechanisms that govern intracellular transport of sterols in eukaryotic cells are not well understood. Saccharomyces cerevisiae is a facultative anaerobic organism that becomes auxotroph for sterols and unsaturated fatty acids in the absence of oxygen. To identify pathways that are required for uptake and transport of sterols, we performed a systematic screen of the yeast deletion mutant collection for genes that are required for growth under anaerobic conditions. Of the approximately 4800 nonessential genes represented in the deletion collection, 37 were essential for growth under anaerobic conditions...
January 2006: Molecular Biology of the Cell
R T Stone, E Casas, T P L Smith, J W Keele, G Harhay, G L Bennett, M Koohmaraie, T L Wheeler, S D Shackelford, W M Snelling
As genetic markers, SNP are well suited for the development of genetic tests for production traits in livestock. They are stable through many generations and can provide direct assessment of individual animal's genetic merit if they are in linkage disequilibrium and phase with functional genetic variation. Bovine chromosome 5 has been shown to harbor genetic variation affecting production traits in multiple cattle populations; thus, this chromosome was targeted for SNP-based marker development and subsequent association analysis with carcass and growth phenotypes...
October 2005: Journal of Animal Science
Martin Bard, Aaron M Sturm, Charles A Pierson, Shaleak Brown, Kristina M Rogers, Sarah Nabinger, James Eckstein, Robert Barbuch, N D Lees, Susan A Howell, Kevin C Hazen
Candida glabrata is emerging as a more common and important human pathogen. It is less susceptible to azole antifungals than Candida albicans, thus, posing some unique treatment challenges. Previously undetected C. glabrata isolates were identified from clinical specimens by adding bile to the growth medium. Cholesterol was found to be the responsible ingredient in bile. Six bile-dependent isolates were characterized and were found to exhibit wild-type equivalent growth when provided human or bovine serum or free cholesterol...
August 2005: Diagnostic Microbiology and Infectious Disease
Lucia Hronská, Zuzana Mrózová, Martin Valachovic, Ivan Hapala
Mild non-ionic detergents are used for solubilization of hydrophobic substrates in yeast growth media at concentrations 0.1-1%. Our data show that low concentrations of Nonidet P-40 may significantly affect lipid biogenesis in the yeast Saccharomyces cerevisiae. The uptake and esterification of external [4-14C]-cholesterol is strongly reduced in hem1 mutants treated with low concentrations of Nonidet P-40. Significant inhibitory effect of NP-40 on sterol uptake and esterification was evident both in non-growing and growing cells supplemented with external cholesterol...
September 1, 2004: FEMS Microbiology Letters
Thierry Ferreira, Matthieu Régnacq, Parissa Alimardani, Carole Moreau-Vauzelle, Thierry Bergès
In the yeast Saccharomyces cerevisiae, UFA (unsaturated fatty acids) and ergosterol syntheses are aerobic processes that require haem. We took advantage of a strain affected in haem synthesis ( hem1 Delta) to starve specifically for one or the other of these essential lipids in order to examine the consequences on the overall lipid composition. Our results demonstrate that reserve lipids (i.e. triacylglycerols and steryl esters) are depleted independently of haem availability and that their UFA and sterol content is not crucial to sustain residual growth under lipid depletion...
March 15, 2004: Biochemical Journal
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