@misc{oh_donofrio_pan_coughlan_brown_meng_mitchell_dean_2008, title={Transcriptome analysis reveals new insight into appressorium formation and function in the rice blast fungus Magnaporthe oryzae}, volume={9}, ISSN={["1474-760X"]}, DOI={10.1186/gb-2008-9-5-r85}, abstractNote={Rice blast disease is caused by the filamentous Ascomycetous fungus Magnaporthe oryzae and results in significant annual rice yield losses worldwide. Infection by this and many other fungal plant pathogens requires the development of a specialized infection cell called an appressorium. The molecular processes regulating appressorium formation are incompletely understood. We analyzed genome-wide gene expression changes during spore germination and appressorium formation on a hydrophobic surface compared to induction by cAMP. During spore germination, 2,154 (approximately 21%) genes showed differential expression, with the majority being up-regulated. During appressorium formation, 357 genes were differentially expressed in response to both stimuli. These genes, which we refer to as appressorium consensus genes, were functionally grouped into Gene Ontology categories. Overall, we found a significant decrease in expression of genes involved in protein synthesis. Conversely, expression of genes associated with protein and amino acid degradation, lipid metabolism, secondary metabolism and cellular transportation exhibited a dramatic increase. We functionally characterized several differentially regulated genes, including a subtilisin protease (SPM1) and a NAD specific glutamate dehydrogenase (Mgd1), by targeted gene disruption. These studies revealed hitherto unknown findings that protein degradation and amino acid metabolism are essential for appressorium formation and subsequent infection. We present the first comprehensive genome-wide transcript profile study and functional analysis of infection structure formation by a fungal plant pathogen. Our data provide novel insight into the underlying molecular mechanisms that will directly benefit efforts to identify fungal pathogenicity factors and aid the development of new disease management strategies.}, number={5}, journal={GENOME BIOLOGY}, author={Oh, Yeonyee and Donofrio, Nicole and Pan, Huaqin and Coughlan, Sean and Brown, Douglas E. and Meng, Shaowu and Mitchell, Thomas and Dean, Ralph A.}, year={2008} }
 @article{donofrio_oh_lundy_pan_brown_jeong_coughlan_mitchell_dean_2006, title={Global gene expression during nitrogen starvation in the rice blast fungus, Magnaporthe grisea}, volume={43}, ISSN={["1087-1845"]}, DOI={10.1016/j.fgb.2006.03.005}, abstractNote={Efficient regulation of nitrogen metabolism likely plays a role in the ability of fungi to exploit ecological niches. To learn about regulation of nitrogen metabolism in the rice blast pathogen Magnaporthe grisea, we undertook a genome-wide analysis of gene expression under nitrogen-limiting conditions. Five hundred and twenty genes showed increased transcript levels at 12 and 48 h after shifting the fungus to media lacking nitrate as a nitrogen source. Thirty-nine of these genes have putative functions in amino acid metabolism and uptake, and include the global nitrogen regulator in M. grisea, NUT1. Evaluation of seven nitrogen starvation-induced genes revealed that all were expressed during rice infection. Targeted gene replacement on one such gene, the vacuolar serine protease, SPM1, resulted in decreased sporulation and appressorial development as well as a greatly attenuated ability to cause disease. Data are discussed in the context of nitrogen metabolism under starvation conditions, as well as conditions potentially encountered during invasive growth in planta.}, number={9}, journal={FUNGAL GENETICS AND BIOLOGY}, author={Donofrio, N. M. and Oh, Y. and Lundy, R. and Pan, H. and Brown, D. E. and Jeong, J. S. and Coughlan, S. and Mitchell, T. K. and Dean, R. A.}, year={2006}, month={Sep}, pages={605–617} }
 @article{thon_pan_diener_papalas_taro_mitchell_dean_2006, title={The  role of transposable element clusters in genome evolution and loss of synteny in the rice blast fungus Magnaporthe oryzae}, volume={7}, number={2}, journal={Genome Biology}, author={Thon, M. R. and Pan, H. Q. and Diener, S. and Papalas, J. and Taro, A. and Mitchell, T. K. and Dean, R. A.}, year={2006} }
 @article{kulkarni_thon_pan_dean_2005, title={Novel G-protein-coupled receptor-like proteins in the plant pathogenic fungus Magnaporthe grisea}, volume={6}, number={3}, journal={Genome Biology}, author={Kulkarni, R. D. and Thon, M. R. and Pan, H. Q. and Dean, R. A.}, year={2005} }
 @article{kudo_bao_a d'souza_ying_pan_roe_canfield_2005, title={The alpha- and beta-subunits of the human UDP-N-acetylglucosamine : lysosomal enzyme phosphotransferase are encoded by a single cDNA}, volume={280}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m509008200}, abstractNote={Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an α2β2γ2-subunit structure was proposed. Although cDNA encoding the γ-subunit has been described, cDNAs for the α- and β-subunits have not. Using partial amino acid sequences from the bovine α- and β-subunits, we have isolated a human cDNA that encodes both the α- and β-subunits. Both subunits contain a single predicted membrane-spanning domain. The α- and β-subunits appear to be generated by a proteolytic cleavage at the Lys928-Asp929 bond. Transfection of 293T cells with the α/β-subunits-precursor cDNA with or without the γ-subunit cDNA results in a 3.6- or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the α-subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of Glc-NAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders. Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an α2β2γ2-subunit structure was proposed. Although cDNA encoding the γ-subunit has been described, cDNAs for the α- and β-subunits have not. Using partial amino acid sequences from the bovine α- and β-subunits, we have isolated a human cDNA that encodes both the α- and β-subunits. Both subunits contain a single predicted membrane-spanning domain. The α- and β-subunits appear to be generated by a proteolytic cleavage at the Lys928-Asp929 bond. Transfection of 293T cells with the α/β-subunits-precursor cDNA with or without the γ-subunit cDNA results in a 3.6- or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the α-subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of Glc-NAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders. In higher eukaryotes most lysosomal hydrolases are targeted to the lysosome via a mannose 6-phosphate (M6P) 2The abbreviations used are:M6Pmannose 6-phosphateBACbacterial artificial chromosomeBLASTNbasic local alignment search tool for nucleotideESTexpressed sequence tagGlcNAc-phosphotransferaseUDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferaseI.M.A.G.E.integrated molecular analysis of genomes and their expressionMIMMendelian inheritance in manMLmucolipidosisRACErapid amplification of cDNA endsTBLASTNtranslated basic local alignment search tool for nucleotidecontiggroup of overlapping clones -dependent pathway. Before targeting, lysosomal enzymes are modified by the addition of M6P in a two-step reaction. In the first step UDP-N-acetylglucosaminelysosomal enzyme phosphotransferase (GlcNAc-phosphotransferase; EC 2.7.8.17) catalyzes the transfer of GlcNAc 1-phosphate from UDP-GlcNAc to the terminal or penultimate mannose on high mannose-type glycans of lysosomal hydrolases (1Reitman M.L. Kornfeld S. J. Biol. Chem. 1981; 256: 11977-11980Abstract Full Text PDF PubMed Google Scholar, 2Reitman M.L. Kornfeld S. J. Biol. Chem. 1981; 256: 4275-4281Abstract Full Text PDF PubMed Google Scholar, 3Waheed A. Hasilik A. von Figura K. J. Biol. Chem. 1982; 257: 12322-12331Abstract Full Text PDF PubMed Google Scholar). The second enzymatic step occurs in the trans-Golgi network, where the covering GlcNAc is removed by N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (EC 3.1.4.45), which has the trivial name “uncovering enzyme” (4Varki A. Kornfeld S. J. Biol. Chem. 1981; 256: 9937-9943Abstract Full Text PDF PubMed Google Scholar, 5Waheed A. Hasilik A. von Figura K. J. Biol. Chem. 1981; 10: 5717-5721Abstract Full Text PDF Google Scholar). The lysosomal enzymes, now modified with M6P, bind to M6P receptors in the trans-Golgi network and are translocated to the endosome and subsequently to the lysosome. Recognition of lysosomal hydrolases by GlcNAc-phosphotransferase is the initiating step in lysosomal hydrolase trafficking. Lysosomal hydrolases that are known substrates for GlcNAc-phosphotransferase exhibit low Km values, whereas non-lysosomal glycoproteins bearing similar glycans have higher Km values (1Reitman M.L. Kornfeld S. J. Biol. Chem. 1981; 256: 11977-11980Abstract Full Text PDF PubMed Google Scholar, 3Waheed A. Hasilik A. von Figura K. J. Biol. Chem. 1982; 257: 12322-12331Abstract Full Text PDF PubMed Google Scholar, 6Bao M. Booth J.L. Elmendorf B.J. Canfield W.M. J. Biol. Chem. 1996; 271: 31446-31451Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). mannose 6-phosphate bacterial artificial chromosome basic local alignment search tool for nucleotide expressed sequence tag UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase integrated molecular analysis of genomes and their expression Mendelian inheritance in man mucolipidosis rapid amplification of cDNA ends translated basic local alignment search tool for nucleotide group of overlapping clones GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography from lactating bovine mammary glands and found to contain three distinct polypeptides. Based on the presence of disulfide-linked α2 and γ2 dimers and quantitative protein sequencing, an α2β2γ2-subunit structure with a molecular mass of 540 kDa was proposed (6Bao M. Booth J.L. Elmendorf B.J. Canfield W.M. J. Biol. Chem. 1996; 271: 31446-31451Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). cDNA and gene encoding the γ-subunit have been isolated and characterized (7Raas-Rothschild A. Cormier-Daire V. Bao M. Genin E. Salomon R. Brewer K. Zeigler M. Mandel H. Toth S. Roe B. Munnich A. Canfield W.M. J. Clin. Investig. 2000; 105: 673-681Crossref PubMed Scopus (144) Google Scholar), but the cDNA(s) or gene(s) for the α- and β-subunits has not been reported. Lysosomal storage diseases are caused by a genetic deficiency of one or more lysosomal enzymes. Enzyme replacement therapy, the therapeutic administration of the missing lysosomal enzyme, is a proven or potential treatment strategy for many lysosomal storage diseases (8Grabowski G.A. Barton N.W. Pastores G. Dambrosia J.M. Banerjee T.K. McKee M.A. Parker C. Schiffmann R. Hill S.C. Brady R.O. Ann. Intern. Med. 1995; 122: 33-39Crossref PubMed Scopus (415) Google Scholar, 9Raben N. Danon M. Gilbert A. Dwivedi S. Collins B. Thurberg B. Mattaliano R. Nagaraju K. Plotz P. Mol. Genet. Metab. 2003; 80: 159-169Crossref PubMed Scopus (181) Google Scholar). The cation-independent M6P receptor is widely expressed on the surface of many cells and can be used for efficient receptor-mediated endocytosis of therapeutic enzymes (10Lobel P. Fujimoto K. Ye R.D. Griffiths G. Kornfeld S. Cell. 1989; 57: 787-796Abstract Full Text PDF PubMed Scopus (170) Google Scholar, 11Canfield W.M. Johnson K.F. Ye R.D. Gregory W. Kornfeld S. J. Biol. Chem. 1991; 266: 5682-5688Abstract Full Text PDF PubMed Google Scholar, 12Jadot M. Canfield W.M. Gregory W. Kornfeld S. J. Biol. Chem. 1992; 267: 11069-11077Abstract Full Text PDF PubMed Google Scholar). However, the enzyme must be modified with sufficient M6P to bind to the receptor with the appropriate affinity. Access to the cDNA(s) for the α- and β-subunits may allow reconstitution of phosphorylation in vitro as well as other strategies to improve the quality of lysosomal enzyme therapeutics. GlcNAc-phosphotransferase is absent or unregulated in a group of diseases of lysosomal targeting termed mucolipidosis (13Hasilik A. Waheed A. von Figura K. Biochem. Biophys. Res. Commun. 1981; 98: 761-767Crossref PubMed Scopus (189) Google Scholar, 14Varki A.P. Reitman M.L. Kornfeld S. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7773-7777Crossref PubMed Scopus (54) Google Scholar, 15Varki A. Reitman M.L. Vannier A. Kornfeld S. Grubb J.H. Sly W.S. Am. J. Hum. Genet. 1982; 34: 717-729PubMed Google Scholar, 16Waheed A. Pohlmann R. Hasilik A. von Figura K. van Elsen A. Leroy J. Biochem. Biophys. Res. Commun. 1982; 105: 1052-1058Crossref PubMed Scopus (107) Google Scholar, 17Reitman M.L. Varki A. Kornfeld S. J. Clin. Investig. 1981; 67: 1574-1579Crossref PubMed Scopus (215) Google Scholar). GlcNAc-phosphotransferase activity is absent in mucolipidosis II (ML II; MIM 252500, I-cell disease), it is reduced in ML IIIA (MIM 252600; pseudo-Hurler polydystrophy), and in ML IIIC (MIM 252605, mucolipidosis III, variant form) it is unregulated. Mutations in the γ-subunit gene have been demonstrated to be the molecular basis of MLIIIC (7Raas-Rothschild A. Cormier-Daire V. Bao M. Genin E. Salomon R. Brewer K. Zeigler M. Mandel H. Toth S. Roe B. Munnich A. Canfield W.M. J. Clin. Investig. 2000; 105: 673-681Crossref PubMed Scopus (144) Google Scholar, 18Raas-Rothschild A. Bargal R. Goldman M. Ben-Asher E. Groener J. Toutain A. Stemmer E. Ben-Neriah Z. Flusser H. Beemer F. Penttinen M. Olender T. Rein A. Bach G. Zeigler M. J. Med. Genet. 2004; 41: 1-5Crossref PubMed Scopus (50) Google Scholar). Because cells from ML IIIC patients retain GlcNAc-phosphotransferase activity, whereas the substrate recognition is unregulated, the catalytic domain of the enzyme is predicted to reside on α- and/or β-subunits of the enzyme. Cloning of the human α- and β-subunit gene will allow us to understand the genetic basis of ML II and ML IIIA. In this paper we describe the cloning of a single cDNA and gene that encodes a precursor of the α- and β-subunits of human GlcNAc-phosphotransferase (gene symbol, GNPTAB). Transfection of the α/β-subunits precursor cDNA with or without co-transfection of the regulatory γ-subunitcDNA resulted in an increase in GlcNAc-phosphotransferase activity on low molecular weight substrate. This demonstrates that the α/β-subunits precursor cDNA encodes the catalytic domain of the enzyme. Oligonucleotides, cDNA Libraries, Clones, Plasmid Vectors—Oligonucleotide primers were synthesized at the Molecular Biology Resource Facility at University Oklahoma Health Sciences Center. Mouse liver Marathon Ready™ cDNA and human whole brain Marathon-Ready™ cDNA were purchased from Clontech (Palo Alto, CA). A human placental cDNA library in λgt10 (catalog 77399) was purchased from ATCC (Manassas, VA). SuperScript™ human brain cDNA library was purchased from Invitrogen. pCR2.1, pUC19, pcDNA 3.1(–), and pcDNA6/V5/His-A were purchased from Invitrogen. Amino Terminal Sequencing of Protein and Peptides—Bovine Glc-NAc-phosphotransferase was isolated as previously described (19Bao M. Booth J.L. Elmendorf B.J. Canfield W.M. J. Biol. Chem. 1996; 271: 31437-31445Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) and subjected to non-reducing SDS-PAGE (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207524) Google Scholar). The gel was either electroblotted onto a polyvinylidene fluoride membrane (21Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar) or subjected to in-gel Lys-C digestion. The protein bands on the polyvinylidene fluoride membrane was stained for 1 min with 0.1% Coomassie Blue in 10% acetic acid, 50% methanol, and excised. Protein bands in the gel were stained, excised, and subjected to in-gel reduction, S-Carboxyamidomethylation, and Lys-C digestion in 0.1 m Tris-HCl, pH 8.0, at 37 °C overnight. The peptides were resolved by reverse phase chromatography on a C18 column equilibrated with 0.1% trifluoroacetic acid and developed with a linear gradient in acetonitrile. Individual peaks were examined by matrix-assisted laser desorption/Ionization-mass spectroscopy to identify a peak containing a single mass. Strategies for peak selection, reverse phase selection, and Edman microsequencing has been described (22Lane W.S. Galat A. Harding M.W. Schreiber S. J. Protein Chem. 1991; 10: 151-160Crossref PubMed Scopus (128) Google Scholar). Proteins on the polyvinylidene fluoride membrane and Lys-C-derived peptides were subjected to automated Edman degradation on an Applied Biosystems, Inc. (Foster City, CA) model 492 protein sequencer at the Molecular Resource Facility in the University of Oklahoma Health Sciences Center. 3′-RACE for the Mouse β-subunit—A primer 5′-gaccgatgaaacaaaaggcaacc-3′ that spans 441–463 nucleotides upstream from the first amino acid of the mouse β-subunit in a mouse cDNA clone (GenBank™ accession number L36434) was used to amplify the 3′-end of the cDNA from mouse liver Marathon-Ready cDNA by nested 3′-RACE. Primers AP1 5′-ccatcctaatacgactcactatagggc-3′ and AP2 5′-actcactatagggctcgagcggc-3′ were used as reverse primers for the first and second PCR, respectively. The cycling parameters for the first PCR were 30 cycles of denaturation at 94 °C for 30 s and annealing/extension at 68 °C for 4 min. The second PCR used the same parameters for 20 cycles. A ∼3.2-kb product was gel-purified, subcloned into pCR2.1, and sequenced on both strands. Screening of Human Placental cDNA Library and 3′-RACE for Human GlcNAc-phosphotransferase cDNA—A 1.1-kb fragment was amplified from the partial mouse β-subunit cDNA by PCR using a forward primer 5′-agtttggttagcccagtgacacc-3′ and a reverse primer 5′-aaatcctgcaggttaaaggtaggtcgtg-3′ and used to screen a size-selected human placental cDNA library in λgt10 under reduced stringency (55 °C, 2× SSC (1× SSC = 0.15 m NaCl and 0.015 m sodium citrate)). Inserts from positive clones were subcloned into pCR2.1 using λgt10 LD-Insert Screening Amplimer Set (Clontech, Palo Alto, CA) and sequenced. The remaining portion of the cDNA was cloned by a combination of a walking strategy and RACE. The 3′-end of the cDNA was amplified from human whole brain Marathon-Ready™ cDNA by nested 3′-RACE, first using primers 5′-agtttggttagcccagtgacacc-3′ and AP1 5′-ccatcctaatacgactcactatagggc-3′, then using primers 5′-cccaaagaaaaacgcttcccgaag-3′ and AP2 5′-actcactatagggctcgagcggc-3′. The cycling parameters for the first PCR were 30 cycles of denaturation at 94 °C for 30 s and annealing/extension at 68 °C for 4 min. The second PCR used the same parameters for 20 cycles. A 3.4-kb product was gel-purified, subcloned into pCR2.1, and sequenced on both strands. Construction of the Full-length cDNA Encoding Human GlcNAc-phosphotransferase α/β-subunits Precursor—A full-length human precursor cDNA was constructed by splicing three pieces of cDNA, the 1118-nucleotide fragment of I.M.A.G.E. Consortium clone 682681 (GenBank™ accession number AA204698), a 1703-nucleotide fragment from the placental clone, and a 2698-nucleotide fragment from the 3′-RACE product by ligation at two BbsI restriction sites and subcloning into pUC19. The full-length cDNA (5.6 kb) was excised and subcloned into the NotI site of pcDNA 3.1(–). Instability of a 5-adenosine repeat (nucleotides 2925–2929) was identified as an occasional problem that results in deletion of 1 adenosine, introducing a frame-shift. This problem was prevented by replacing a 215-bp nucleotide fragment between the MfeI and DraIII sites with an MfeI- and DraIII-cleaved PCR product. The product was amplified by using the cDNA as a template with a forward superprimer 5′-gaagacacaattggcatacttcactgatagcaagaatactgggaggcaactaaaagatac-3′ and a reverse primer 5′-actgcatatcctcagaatgg-3′. The sequence aaaaa was replaced by aagaa (underlined in the forward primer sequence) that does not alter the amino acid sequence. Construction of Expression Plasmids for the Precursor—To generate expression plasmids for the precursor, the following modifications were made. The 3′-untranslated region was removed by further subcloning an ∼3.8-kb NheI-BglII fragment from the cDNA between NheI and BamHI sites of pcDNA6/V5/His-A. The sequence around the initiation codon was modified in an attempt to improve expression. A ∼1.1-kb fragment between the NheI site in the polylinker and the XhoI site in the cDNA was replaced by NheI- and XhoI-cleaved PCR products amplified using various forward primers described below and a reverse primer, 5′-ctaaaggtaggcaagtggctc-3′. Forward primers were 5′-cgtggcgggctagccaccatgxyzttcaagctcctgcagagaca-3′, where xyz is ctg, ggg, gcg, and gtg, generating a second amino acid of Leu, Gly, Ala, and Val, respectively. Screening of a Human Genomic BAC Library—A 107-bp fragment was amplified by PCR from a SuperScript™ human brain cDNA library using primers 5′-tgcagagacagacctatacctgcc-3′ and 5′-actcacctctccgaactggaaag-3′. The cycling parameters were 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 79 °C for 1 min. The product was used as a probe for screening a human genomic BAC library by Genome Systems (St. Louis, MO). Four genomic clones were identified, and BAC 14951 was isolated and sequenced as described previously (23Pan H. Wang Y. Chissoe S. Bodenteich A. Wang Z. Iyer K. Clifton S. Crabtree J. Roe B. Genet. Anal. Tech. Appl. 1994; 11: 181-186Crossref PubMed Scopus (33) Google Scholar, 24Bodenteich A. Chissoe S. Wang Y. Roe B. Venter J. Automated DNA Sequencing and Analysis Techniques. Academic Press, Ltd., London1993: 42-50Google Scholar, 25Chissoe S.L. Bodenteich A. Wang Y.-F. Wang Y.-P. Burian D. Clifton S.W. Crabtree J. Freeman A. Iyer K. Jian L. Ma Y. McLaury H.-J. Pan H.-Q. Sharan O. Toth S. Wang Z. Zhang G. Heisterkamp N. Groffen J. Roe B.A. Genomics. 1995; 27: 67-82Crossref PubMed Scopus (174) Google Scholar). Briefly, fragments in the 1–3-kbp range were randomly sheared from the purified BAC DNA (50 μg), blunt-ended, and subcloned into SmaI site of pUC18. A random library was generated by transforming Escherichia coli strain XL1BlueMRF′ (Stratagene, La Jolla, CA) by electroporation. ∼1200 colonies were picked from each transformation, and the sequencing templates were isolated by a cleared lysate-based protocol from the cultured library (24Bodenteich A. Chissoe S. Wang Y. Roe B. Venter J. Automated DNA Sequencing and Analysis Techniques. Academic Press, Ltd., London1993: 42-50Google Scholar). Sequencing was performed as previously described (25Chissoe S.L. Bodenteich A. Wang Y.-F. Wang Y.-P. Burian D. Clifton S.W. Crabtree J. Freeman A. Iyer K. Jian L. Ma Y. McLaury H.-J. Pan H.-Q. Sharan O. Toth S. Wang Z. Zhang G. Heisterkamp N. Groffen J. Roe B.A. Genomics. 1995; 27: 67-82Crossref PubMed Scopus (174) Google Scholar). After base-calling with the ABI analysis software, the analyzed data were transferred to a Sun workstation cluster for base-calling and assembly using Phred and Phrap programs (26Ewing B. Hillier L. Wendle M. Green P. Genome Res. 1998; 8: 175-185Crossref PubMed Google Scholar, 27Ewing B. Green P. Genome Res. 1998; 8: 186-194Crossref PubMed Scopus (4901) Google Scholar), respectively. Overlapping sequences and contigs were analyzed using Consed (28Gordon D. Abajian C. Green P. Genome Res. 1998; 8: 195-202Crossref PubMed Scopus (2854) Google Scholar). Gap closure and proofreading was performed using either custom primer-walking or PCR amplification of the region corresponding to the gap in the sequence followed by subcloning into pUC18 and cycle-sequencing with the universal pUC primers. In some instances additional synthetic custom primers were necessary for at least 3-fold coverage for each base. Northern Blot Analysis—A cDNA fragment corresponding to nucleotides 1252–3356 of the cDNA was excised by XhoI and EcoRV from the plasmid containing the full-length cDNA. The cDNA was labeled with [γ-32P]dCTP using random hexamer priming with a Klenow fragment of DNA polymerase I (Readiprime II random prime labeling System, Amersham Biosciences) and used to probe a human MTN™ (multi-tissue northern) blot from BD Bioscience following the manufacturer's recommendation, except that all the washing steps were performed at 65 °C. The membrane was exposed to a BIOMAX MS x-ray film (Eastman Kodak Co.) with an intensifying screen for 16 h at –80 °C. Expression of the Precursor in 293T Cells—The human embryonic kidney cell line 293T was grown in Dulbecco's modified Eagle's medium with 10% (v/v) fetal bovine serum at 37 °C and 5% CO2, 95% air. Cells were transfected with empty vector or vector containing cDNA using FuGene-6 (Roche Applied Science). Cells were harvested by scraping at 72 h post-transfection and incubated in lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 2 mm MgCl2) on ice for 30 min (14Varki A.P. Reitman M.L. Kornfeld S. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7773-7777Crossref PubMed Scopus (54) Google Scholar). The lysate was clarified by centrifugation at 40,000 × g for 20 min, and the supernatant was assayed for GlcNAc-phosphotransferase activity as described below and protein concentration by Bradford assay (Pierce). GlcNAc-phosphotransferase Assay—GlcNAc-phosphate transferred to α-methylmannoside was determined using [β-32P]UDP-GlcNAc as described previously (29Reitman M.L. Lang L. Kornfeld S. Methods Enzymol. 1984; 107: 163-172Crossref PubMed Scopus (24) Google Scholar). One unit of GlcNAc-phosphotransferase activity is defined as 1 pmol of GlcNAc phosphate transferred to α-methylmannoside/h in a reaction containing 150 μm UDP-GlcNAc and 100 mm α-methylmannoside. Alignments of Deduced Protein Sequences—GlcNAc-phosphotransferase sequences derived from databases were assembled and translated. Deduced protein sequences were aligned using ClustalW (30Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55998) Google Scholar) as implemented by MacVector™ 7.2 (Accelrys, San Diego, CA) Bovine GlcNAc-phosphotransferase Protein Sequence—Bovine Glc-NAc-phosphotransferase was purified 488,000-fold to apparent homogeneity as previously described (19Bao M. Booth J.L. Elmendorf B.J. Canfield W.M. J. Biol. Chem. 1996; 271: 31437-31445Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), subjected to non-reduced SDS-PAGE, and transferred to a polyvinylidene fluoride membrane. The bands corresponding to the α-subunit dimer (320 kDa) and β-subunit (56 kDa) were excised and subjected to amino-terminal sequencing. Duplicate samples were subjected to in situ Lys-C digestion and fractionated by reverse phase high performance liquid chromatography, and several isolated peptides were subjected to amino-terminal microsequencing. The α-subunit bands generated a single amino-terminal sequence of MLLKLLQRQTY and internal peptide sequences of VPMLVLDXAXPTXVXLK and DLPSLYPSFLSASDVFNVAKPK. The β-subunit bands generated an amino-terminal sequence of DTFADSLRYVNKILNSKFGFTSRKVPAH and internal peptide sequences of ILNSK, TSFHK, FGFTSR, and SLVTNCKPVTDK. Two of the internal sequences, ILNSK and FGFTSR, overlap with the amino-terminal sequence. Molecular Cloning of a Partial Mouse GlcNAc-phosphotransferase β-subunit cDNA—Bovine GlcNAc-phosphotransferase β-subunit peptide sequences were used to search the EST data base using the TBLASTN algorithm (31Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71439) Google Scholar). The search identified a mouse cDNA (GenBank™ accession number L36434) that was previously incorrectly annotated as a basic domain-leucine zipper transcription factor (32Cordes S.P. Barsh G.S. Cell. 1994; 79: 1025-1034Abstract Full Text PDF PubMed Scopus (340) Google Scholar). The 1846-bp murine clone contained highly homologous sequences to the determined β-subunit amino-terminal and internal peptide amino acid sequences. The amino-terminal sequence of the bovine β-subunit began at nucleotide 799 of the mouse cDNA. The 798-bp sequence 5′ to this revealed an in-frame open reading frame that lacked evidence for either a signal peptide or an initiator methionine, suggesting the β-subunit might be derived from a larger precursor. The murine clone was extended by 3′-RACE as described under “Experimental Procedures,” yielding a ∼4600-bp partial cDNA. Attempts to extend the 5′-sequence by 5′-RACE, library screening, and data base searching were unsuccessful. Molecular Cloning and Characterization of Human GlcNAc-phosphotransferase α- and β-subunits Precursor cDNA—A 1.1-kb fragment from the mouse cDNA was used to screen a human placental cDNA library in λgt10 under reduced stringency. Several positive clones were isolated, and the clone with the largest insert (3.4 kb) was sequenced. The remaining portion of the cDNA was cloned by a combination of a walking strategy, 3′-RACE, and data base searching. The placental sequence was used to design nested 3′-RACE primers that allowed the isolation of a 3′-RACE product from human brain Marathon-Ready™ cDNA. The 3426-nucleotide 3′-RACE product contained a 1761-nucleotide coding region and a 1665-nucleotide 3′-untranslated region. The placental sequence was also used to search the EST data base using the BLASTN algorithm (31Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71439) Google Scholar). The search identified an I.M.A.G.E. clone 682681 (GenBank™ Accession Number AA204698) which contained a 1271-bp insert composed of a 164-nucleotide 5′-untranslated region and 1106-nucleotide coding region that overlapped the 5′ portion of the placental sequence. Together, these strategies allowed the isolation of a full-length cDNA that spanned 5597 bp. The cDNA sequence for human GlcNAc-phosphotransferase has been deposited to GenBank™ and the accession number is AY687932. Deduced Structure of GlcNAc-phosphotransferase α/β-subunits Precursor—The nucleotide and deduced amino acid sequences of human GlcNAc-phosphotransferase α/β-subunits precursor are shown in Fig. 1. The 5597-bp cDNA is predicted to encode a protein of 1256 amino acids. The cDNA appears to encode an precursor with the α-subunit in the 5′-position and the β-subunit in the 3′-position. Sequences highly homologous to all the amino-terminal and internal-peptide sequences of both the α- and β-subunit of bovine GlcNAc-phosphotransferase are represented in the predicted human protein sequence (89 and 100% identity in the α- and β-subunits, respectively). The precursor protein has a predicted molecular mass of 143,582 Da, of which 104,706 and 38,894 Da are for the α- and β-subunits, respectively. The sequence contains 17 consensus sites for N-glycosylation in the α-subunit and 3 in the β-subunit. It also contains 22 cysteine residues, 18 in the α-subunit and 4 in the β-subunit.FIGURE 1Nucleotide and deduced amino acid sequences of the human GlcNAc-phosphotransferase α/β-subunits precursor. The predicted protein sequence is shown below the DNA sequence. The putative transmembrane domains are underlined. Sequences homologous to the experimentally identified bovine α- and β-subunits amino acid sequences are indicated by dashed underlines. The amino terminus of the β-subunit is indicated by an arrow. The 20 potential sites for N-linked glycosylation are indicated by asterisks. An in-frame stop codon upstream of the initiation codon is double-underlined. A potential polyadenylation signal is shown by dotted underline.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The sequence surrounding the proposed initiation codon (ggggtgatgc) has a guanosine in position –3 and a cytidine in position +4, yielding a non-preferred translation initiation sequence (33Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4172) Google Scholar). There is an in-frame stop codon 129 nucleotides upstream from the initiation codon. The cDNA has a single potential polyadenylation signal (ATAAAA) 13–18 nucleotides upstream from the poly(A) tail, although the signal was not the more commonly found AATAAA or ATTAAA. Membrane Topology of GlcNAc-phosphotransferase α- and β-subunits—Examination of a Kyte-Doolittle hydrophilicity plot (34Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17289) Google Scholar) generated by MacVector™ 7.2 (Accelrys, San Diego, CA) of the predicted GlcNAc-phosphotransferase precursor sequence (Fig. 2) reveals prominent hydrophobic segments of 24 and 26 residues near the amino terminus and the carboxyl terminus, respectively. Because the amino terminus of the deduced human α-subunit sequence was similar to the amino terminus}, number={43}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Kudo, M and Bao, M and A D'Souza and Ying, F and Pan, HQ and Roe, BA and Canfield, WM}, year={2005}, month={Oct}, pages={36141–36149} }
 @article{dean_talbot_ebbole_farman_mitchell_orbach_thon_kulkarni_xu_pan_et al._2005, title={The genome sequence of the rice blast fungus Magnaporthe grisea}, volume={434}, ISSN={["1476-4687"]}, DOI={10.1038/nature03449}, abstractNote={Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft sequence of the M. grisea genome. Analysis of the gene set provides an insight into the adaptations required by a fungus to cause disease. The genome encodes a large and diverse set of secreted proteins, including those defined by unusual carbohydrate-binding domains. This fungus also possesses an expanded family of G-protein-coupled receptors, several new virulence-associated genes and large suites of enzymes involved in secondary metabolism. Consistent with a role in fungal pathogenesis, the expression of several of these genes is upregulated during the early stages of infection-related development. The M. grisea genome has been subject to invasion and proliferation of active transposable elements, reflecting the clonal nature of this fungus imposed by widespread rice cultivation.}, number={7036}, journal={NATURE}, author={Dean, RA and Talbot, NJ and Ebbole, DJ and Farman, ML and Mitchell, TK and Orbach, MJ and Thon, M and Kulkarni, R and Xu, JR and Pan, HQ and et al.}, year={2005}, month={Apr}, pages={980–986} }
 @article{ebbole_jin_thon_pan_bhattarai_thomas_dean_2004, title={Gene discovery and gene expression in the rice blast fungus, Magnaporthe grisea: Analysis of expressed sequence tags}, volume={17}, ISSN={["0894-0282"]}, DOI={10.1094/MPMI.2004.17.12.1337}, abstractNote={Over 28,000 expressed sequence tags (ESTs) were produced from cDNA libraries representing a variety of growth conditions and cell types. Several Magnaporthe grisea strains were used to produce the libraries, including a nonpathogenic strain bearing a mutation in the PMK1 mitogen-activated protein kinase. Approximately 23,000 of the ESTs could be clustered into 3,050 contigs, leaving 5,127 singleton sequences. The estimate of 8,177 unique sequences indicates that over half of the genes of the fungus are represented in the ESTs. Analysis of EST frequency reveals growth and cell type-specific patterns of gene expression. This analysis establishes criteria for identification of fungal genes involved in pathogenesis. A large fraction of the genes represented by ESTs have no known function or described homologs. Manual annotation of the most abundant cDNAs with no known homologs allowed us to identify a family of metallothionein proteins present in M. grisea, Neurospora crassa, and Fusarium graminearum. In addition, multiply represented ESTs permitted the identification of alternatively spliced mRNA species. Alternative splicing was rare, and in most cases, the alternate mRNA forms were unspliced, although alternative 5' splice sites were also observed.}, number={12}, journal={MOLECULAR PLANT-MICROBE INTERACTIONS}, author={Ebbole, DJ and Jin, Y and Thon, M and Pan, HQ and Bhattarai, E and Thomas, T and Dean, R}, year={2004}, month={Dec}, pages={1337–1347} }