摘要
作为供给人体叶酸需求的主要来源之一,植物中的主要作物如水稻、小麦和玉米的叶酸含量很低。对植物叶酸合成途径调控机理的研究,目前仅对GTP环化脱羧酶(GCHI)有较深入的研究。本文拟对植物叶酸生物合成途径中的另外2个酶即二氢叶酸合成酶/多谷氨酰胺叶酸合成酶(DHFS/FPGS)以及二氢喋呤合成酶(DHPS)进行研究,探讨它们在植物(拟南芥)叶酸生物合成中的作用。在植物中DHFS和FPGS分别由不同的基因编码,而在E.coli中则由FolC基因同时编码;DHPS在E.coli中由FolP基因编码,在植物中则由类似的基因编码。将来源于E.coli的FolC和FolP基因分别与酵母CoxⅣ基因的线粒体定位序列连接,并由CaMV 35S启动子驱动。取得了以下主要研究结果:
1.系统地分析了来源于E.coli的FolC基因的作用:(1) Northern blot和Taqman RT-PCR分析表明,来源于E.coli的FolC基因在拟南芥植株中得到了表达。(2) 对FolC转基因纯系C4-3-3、C11-6-8和C12-13-1的幼苗植株和幼嫩叶片的叶酸含量分析表明,这3个纯系幼苗植株的叶酸含量分别为2.23、2.46和2.51 nmol/g鲜重,而对照为1.77 nmol/g鲜重:这3个纯系其幼嫩叶片的叶酸含量分别是5.07、5.54和4.31 nmol/g鲜重,对照为3.09 nmol/g鲜重。因此无论是幼苗植株还是幼嫩叶片,转基因纯系的叶酸含量均显著高于对照。(3) mRNA表达高的纯系C11-6-8,其叶酸含量也高,而mRNA表达较C11-6-8低的纯系C4-3-3和C12-13-1,则其叶酸含量也相应低,因此FolC转基因纯系的叶酸含量与FolC基因的mRNA表达量呈正相关。(4) 对转基因纯系其内源DHFS/FPGS编码基因的mRNA表达的定量分析结果表明,外源FolC基因在幼嫩叶片中的过量表达,并没有促进内源DHFS/FPGS编码基因的表达。
因此外源FolC基因在拟南芥植株中的过量表达,提高了植株的叶酸含量,而且转基因植株叶酸含量的提高是由于外源FolC基因而非内源DHFS/FPGS编码基因作用的结果。
2.分析了拟南芥野生型植株内源DHFS/FPGS编码基因的表达以及植株不同发育时期叶片的叶酸含量。Taqman RT-PCR分析表明:内源不同的DHFS/FPGS编码基因其mRNA表达水平不同,幼嫩叶片中DHFS和FPGS-3的表达量最高;此外,DHFS和FPGS-1、FPGS-2、FPGS-3的总表达量在幼嫩叶片中最高,具有组织特异性。叶酸的测定结果表明,不同发育时期叶片的叶酸含量不同。结合内源DHFS/FPGS编码基因的表达分析以及对叶酸含量的分析,结果显示:野生型植株的叶酸含量与内源DHFS/FPGS编码基因的mRNA表达量呈正相关。
3.FolC转基因植株比野生型植株开花明显提前,但对植株的生长发育没有负面影响,相反FolC单拷贝纯系C11-6-1和C12-13-1的单株结荚数和单株干重还明显高于对照植株。
4.Northern blot分析表明,来源于E.coli的FolP基因在拟南芥植株中得到了表达。对FolP转基系株系的叶酸测定结果显示,独立株系P26、P45和P49的T_3幼苗其叶酸含量分别是2.30、1.63和2.03 nmol/g鲜重,而野生型对照为1.32 nmol/g鲜重,转基因株系的叶酸含量均显著高于对照。
综上所述,本文首次研究了DHFS/FPGS和DHPS的编码基因对提高拟南芥叶酸合成的作
Unlike plants and microorganisms, humans can not synthesize folate de novo and depend entirely on their dietary supply. Plant foods are the single most important source of folate. However many plant food sources such as rice, wheat and maize are poor in this vitamin.Though the folate biosynthesis pathway of plants is now largely elucidated, little is known about the control of flux and accumulation of folate. The first two pioneering trails of the regulation of folate biosynthesis foucued on GTP cyclohydrolase I (GCHI). The aim of this study is to elucicate whether the other two enzymes of the folate biosynthesis pathway , i.e. dihydrofolate synthase(DHFS)/folypolyglutamate synthase(FPGS) and dihydropteroate synthase (DHPS) will increased the folate content in A. thaliana through genetic modification. DHFS/FPGS and DHPS in E.coli was encoded by FolC and FolP gene, respectively. Each of FolC and FolP gene was fused to the mitochondria targeting sequence from yeast CoXIV gene, and was promoted by double CaMV 35S, respectively. The main results obtained as follows:1. Systematically study of the FolC gene from E.coli was carried out on transgenic Arabidopsis: (1) The analysis by Northern blot and Taqman RT-PCR showed that the FolC gene from E.coli was expressed in Arabidopsis. (2) Folate measurement was conducted both on seedlings and young green leaves. In seedlings, the folate content of homozygous FolC transgenic lines C4-3-3 、 C11-6-8 and C12-13-1 was 2.23, 2.46 and 2.51 nmol /gfw, respectively, and that of the wild type plants was 1.77 nmol /gfw. While in young green leaves , the folate content was 5.07, 5.54 and 4.31 nmol/gfw, respectively, and that of the wild type plants was 3.09 nmol /gfw. Therefore the folate content of the transgenic line was significantly higher than that of wild type plants both in seedlings and young green leaves. (3) The highest expresser of FolC (C11-6-8) also showed the highest folate content, while line C4-3-3 and C12-13-1, which the expression of FolC was lower than that of C11-6-8, showed a relatively lower folate content. It was thun indicated the mRNA level of FolC gene was correlated with the folate level of FolC transgenic lines. (4) The overexpression of FolC gene did not promote the expression of the endogenous genes encoding DHFS/FPGS in FolC transgenic lines.Therefore the overexpression of foreign FolC gene resulted in the enhancement of folate content in Arabidopsis, and it was proved that the increase folate content was from the contribution of foreign FolC gene but not the endogenous genes encoding DHFS/FPGS .2. The profiling of the expression of the endogenous genes encoding DHFS/FPGS was conducted on
引文
1. Achari A, Somers DD, Champness JN, Bryant PK, Rosemond J, Stammers DK. Crystal structure of the anti-bacterial sulfonamide drug target dihydropteroate synthase. Nat. Struct. Biol. 1997, 4: 490-497.
2. Agius F, Gonzalez-Lamothe R, Caballero JL, Munoz-Blanco J, Botella MA, Valpuesta V. Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase. Nat. Biotechnol. 2003, 21: 134-136.
3. Allegra CJ, Boarman D, Kovacs JA, Morrison P, Beaver J, Chabner BA, Masur H. Primary structure and expression of the dihydropteroate synthase gene of Plasmodium falciparum. J. Clin. Invest. 1990,85:371-379.
4. Andrew DH, Jesse FG Synthesis and turnover of folates in plants Current Opinion in Plant Biology 2002, 5: 244-249.
5. Antony AC. Folate receptors. Ann. Rev. Nutrn. 1996,16:501-521.
6. Appling DR. Compartmentation of folate-mediated one-carbon metabolism in eukaryotes. FASEBJ. 1991,5:2645-2651.
7. Auerbach G, Herrmann A, Bracher A, Bader G, Gutlich M, Fischer M, Neukamm M, Garrido-Franco M, Richardson J, Nar H et al. Zinc plays a key role in human and bacterial GTP cyclohydrolase I. Proc. Natl. Acad. Sci. USA, 2000,97: 13567-13572.
8. Aymeric G, Victoria I, Sanja R, Markus F, Adelbert B, Andrew DH. Folate Biosynthesis in Higher Plants. cDNA Cloning, Heterologous Expression, and Characterization of Dihydroneopterin Aldolases. Plant Physiology, 2004, 135: 103-111.
9. Bagley PJ, Selhub J. Analysis of folate form distribution by affinity followed by reversed-phase chromatography with electrochemical detection. Clin. Chem. 2000,46: 404-411.
10. Bagley PJ, Selhub J. Analysis of folates using combined affinity and ion-pair chromatography. Methods Enzymol. 1997,281:16-25.
11. Bailey LB, Moyers S, Gregory JF. Folate. In Present Knowledge in Nutrition, edn 8. Edited by Bowman BA, Russell RM. Washington DC, ILSI Press, 2001: 214-229.
12. Bak S, Olsen CE, Petersen BL, Lindberg MB, Halkier BA. Metabolic engineering of p-hydroxybenzylglucosinolate in Arabidopsis by expression of the cyanogenic CYP79A1 from Sorhghum bicolor. Plant J. 1999, 20: 663-671.
13. Basset G, Quinlivan EP, Ziemak MJ, Diaz GR, Fischer M, Schiffrnann S, Bacher A, Gregory JF, Hanson AD. Folate synthesis in plants: the first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I. Proc. Natl. Acad. Sci. USA, 2002, 99: 12489-12494.
14. Basset GJC, Quinlivan EP, Ravanel S, Rebeille F, Nichols BP, Shinozaki K, Seki M, Adams-Phillips LC, Giovannoni JJ, Gregory JF, Hanson AD. Folate synthesis in plants: The p-aminobenzoate branch is initiated by a bifunctional PabA-PabB protein that is targeted to plastids. Proc. Natl. Acad. Sci. USA, 2004.
15. Berry RJ, Li Z, Erickson JD, Li S, Moore CA, Wang H, Mulinare J, Zhao P, Wong LY, Gindler J, Hong, SX, Correa A. Prevention of neural tube defects with folic acid in China. N. Engl. J. Med. 1999,341: 1485-1490.
16. Beverley SM, Ellenberg TE, Cordingley JS. Primary structure of the gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase of Leishmania major. Proc. Natl. Acad. Sci. 1986, 83: 2584-2588.
17. Bhandari SD, Gregory JF. Folic acid, 5-methyl-tetrahydrofolate and 5-formyl-tetrahydrofolate exhibit equivalent intestinal absorption, metabolism and in vivo kinetics in rats. Journal of Nutrition 1992, 122: 1847-1854.
18. Black MT, Meyer D, Widge WR, Cramer WA. Light regulated methylation of chloroplast proteins. J. Biol. Chem. 1987,262: 9803-9807.
19. Blakley RL. Dihydrofolate reductase, in Folates and Pterins Vol 1, eds Blakley RL and Benkovic S. J Wiley (Interscience), New York, 1984, ppl91-253.
20. Blaszczyk J, Shi G, Yan H, Ji X. Catalytic center assembly of HPPK as revealed by the crystal structure of a ternary complex at 1.25 AI resolution. Structure, 2000, 8: 1049-1058.
21. Blount BC, Mack MM, Wehr CM, MacGregor JT, Hiatt RA, Wang G, Wickramasinghe SN, Everson RB, Ames BN. Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage. Proc. Natl. Acad. Sci. USA, 1997, 94: 3290-3295.
22. Bognar AL, Osborne C, Shane B, Singer SC, Ferone R. Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product. J. Biol. Chem. 1985, 260: 5625-5630.
23. Botto LD, Moore CA, Khoury MJ, Erickson JD. Neural tube defects. N. Engl. J. Med. 1999, 341: 1509-1510.
24. Bourguignon J, Rebeille F, Douce R. Serine and glycine metabolism in higher plants, in Plant Amino Acids. Ed by Singh B. Marcel Dekker, Inc, New York, 1999, pp111-146.
25. Bovy A, Vos R, Kemper M, Schijlen E, Almenar-Pertejo M, Muir S, Collins G, Robinson S, Verhoeyen M, Hughes S, Santos-Buelga C, Tunen A. High-avonol tomatoes resulting from heterologous expression of the maize transcription factor gene LC and C1. Plant Cell. 2002, 14: 2509-2526.
26. Bree A, Dusseldorp M, Brouwer LA, Hof KH, Steegers-Theunissen RPM. Folate intake in Europe: recommended, actual and desired intake. Eur. J. Clin. Nutr. 1997, 51: 643-60.
27. Bronstrup A, Hages M, Prinz-Langenohl R, Pietrzik K. Effects of folic acid and combinations of folic acid and vitamin B-12 on plasma homocysteine concentrations in healthy, young men. Am. J. Clin. Nutr. 1998, 68: 1104-1110.
28. Brouwer IA, Van Dusseldorp M, Thomas C, Duran M, Hautvast J, Eskes T, Steegers-Theunissen R. Low-dose folic acid supplementation decreases plasma homocysteine concentrations: a randomized trial. Am. J. Clin. Nutr. 1999, 69: 99-104.
29. Cahoon EB, Hall SE, Ripp KG, Ganzke TS, Hitz WD, Coughlan SJ. Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content. Nat. Biotechnol. 2003, 9: 1082-1087.
30. Castle, WB. Megaloblastic anemia. Postgrad. Med. 1978,64: 117-122.
31. Centers for Disease Control and Prevention. Morbid. Mortal. Wkly. Rep. 1996,44 (SS-4): 1-13.
32. Centers for Disease Control and Prevention. Recommendations for the use of folic acid to reduce the number of cases of spina bifida and other neural tube defects. MMWR Recomm. Rep. 1992, 41: 1-7.
33. Chen L, Chan S, Cossins EA. Distribution of folate derivatives and enzymes for synthesis of 10-formyltetrahydrofolate in cytosolic and mitochondrial fractions of pea leaves. Plant Physiol. 1997,115:299-309.
34. Chen MJ, Shimada T, Moulton AD, Cline A, Humphries RK, Maizel J, Nienhuis AW. The functional human dihydrofolate reductase gene. J. Biol. Chem. 1984, 259: 3933-3943.
35. Chen Z, Young TE, Ling J, Chang S, Gallie DR. Proc. Natl. Acad. Sci. USA, 2003, 100: 3525-3530.
36. Cherest H, Thomas D, Surdin-Kerjan YJ. Polyglutamylation of folate coenzymes is necessary for methionine biosynthesis and maintenance of intact mitochondrial genome in Saccharomyces cerevisiae.Biol. Chem. 2000,275: 14056-14063.
37. Choi SW, Mason JB. Folate status: effects on pathways of colorectal carcinogenesis. J. Nutr. 2002, 132:2413S-2418S.
38. Clandinin MT, Cossins EA. Localization and interconversion of tetrahydropteroylpolyglutamates in isolated pea mitochondria. Biochem J. 1972,128: 29-40.
39. Clifford AJ, Heid MK, Peerson JM, Bills ND. Bioavailability of food folates and evaluation of food matrix effects with a rat bioassay. J. Nutr. 1991,121: 445-453.
40. Cossins EA, Chan PY. Folypolyglutamate synthetase activities of Neurospora. Phytochemistry. 1984,23:965-971.
41. Cossins EA, Chen L. Folate and one-carbon metabolism in plants and fungi. Phytochemistry, 1997,45:437-452.
42. Cossins EA, Shah SPJ. Pteroylglutamates of higher plant tissues. Phytochemistry, 1972, 11: 587-593.
43. Cossins EA. The fascinating world of folate and one-carbon metabolism. Can. J. Bot. 2000, 78: 691-708.
44. Cragan JD, Roberts HE, Edmonds LD, Khoury MJ, Kirby RS, Shaw GM, Velie EM, Merz RD, Forrester MB, Williamson RA, Krishnamurti DS, Stevenson RE, Dean JH. Surveillance for anencephaly and spina bifida and the impact of prenatal diagnosis: United States, 1985-1994. Morb. Mortal. Wkly. Rep. CDC Surveill. Summ. 1995,44: 1-13.
45. Crouse GF, Simonsen CC, McEwan RN, Schimke RT. Structure of amplied normal and variant dihydrofolate reductase genes in mouse sarcoma S180 cells. J. Biol. Chem. 1982, 257: 7887-7897.
46. Cybulski RL, Fisher RR. Uptake of oxidized folates by rat liver mitochondria. Biochim. Biophys. Acta. 1981,646:329-333.
47. Czeizel AE. Primary prevention of neural-tube defects and some other major congenital abnormalities: recommendations for the appropriate use of folic acid during preganancy. Paediatr. Drugs. 2000,2: 437-449.
48. Dallas WS, Gowen J, Ray PH, Cox J, Dev IK. Cloning, sequencing, and enhanced expression of the dihydropteroate synthase gene of Escherichia coli MC4100. J. Bacteriol. 1992, 174: 5961-5970.
49. David S, Dean DP. Elevating the Vitamin E Content of Plants Through Metabolic Engineering. Science, 1998, 282: 2098-2100.
50. Derek JM, Kristina P, Donna D, Joseph MM, Joan HJG, Villota R. Folates in foods: reactivity, stability during during processing and nutritional implications. CRC Critical Reviews in Food Science and Nutrition, 1989,28: 438-538.
51. DeSouza L, Shen Y, Bognar AL. Disruption of cytoplasmic and mitochondrial folylpolyglutamate synthetase activity in Saccharomyces cerevisiae. Arch. Biochem. Biophys. 2000,376:299-312.
52. Dixon RA. Natural products and plant disease resistance. Nature, 2001,411: 843-847.
53. Duch DS, Browns SW, Wolf JH, Nichol CA. Biopterin cofactor biosynthesis: GTP cyclohydrolase, neopterin and biopterin in tissues and body fluids of mammalian species. Life Sci. 1984,35: 1895-1901.
54. Erik JMK, Harry HSR, Elisabeth D, Alexandra G, Wim HMS, Piet AB. Folate intake of the Dutch population according to newly established liquid chromatography data for foods. Am. J. Clin.Nutr. 2001, 73: 765-76
55. Finglas PM, Wigertz K, Vahteristo L, Witthoft C, Southon S, Froidmont-Gortz I. Standardisation of HPLC techniques for the determination of naturally-occuring folates in food. Food Chemistry, 1999, 64: 245-255.
56. Fishman, SM, Christian P, West KP. The role of vitamins in the prevention and control of anemia. Public Health Nutr. 2000, 3: 125-150.
57. Fraser PD, Romer S, Shipton CA, Mills PB, Kiano JW, Misawa N,. Drake RG, Schuch W, Bramley PM. Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruitspecific manner. Proc. Natl. Acad. Sci. USA, 2002, 99:1092-1097.
58. Freemantle SJ, Taylor SM, Krystal G, Moran RG Upstream organization of and multiple transcripts from the human folylpoly-gamma-glutamate synthetase gene.J. Biol. Chem. 1995, 270: 9579-9584.
59. Gambonnet B, Jabrin S, Ravanel S, Karan M, Douce R, Rebeille F. Folate distribution during higher plant development. J. Sci. Food Agric. 2001, 81:835-841.
60. Geiger M, Badur R, Kunze I, Sommer S. Changing the fine chemical content in organisms by genetically modifying the shikimate pathway. WO 02/00901 (SunGene), 2001.
61. Geiger M, Badur R. Erhohung des Vitamingehaltes in Organismen durch Erhohung der Tyrosin Aminotransferase Aktivitat. DE 10111676 (SunGene), 2001.
62. Gilles B, Eoin PQ, Michael JZ, Rocyo DG, Markus F, Susi S, Adelbert B, Jesse FGIII, Hanson AD. Folate synthesis in plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I. Proc. Natl. Acad. Sci. USA, 2002, 99: 12489-12494.
63. Giovannucci E, Rimm EB, Ascherio A, Stampfer MJ, Colditz GA, Willett WC. Alcohol low-methionine-low-folate diets, and risk of colon cancer in men. J. Natl. Cancer Inst. 1995, 87: 265-273.
64. Giovannucci E. Epidemiologic Studies of Folate and Colorectal Neoplasia: a Review. J. Nutr. 2002,132: 2350S-2355S.
65. Glynn SA, Albanes D, Pietinen P, Brown CC, Rautalahti M, Tangrea JA, Gunter EW, Barrett MJ, Virtamo J, Taylor PR. Colorectal cancer and folate status: a nested casecontrol study among male smokers. Cancer Epidemiol. Biomarkers Prev. 1996, 5: 487-494.
66. Goli DM, Vanderslice JT. Investigation of the conjugase treatment procedure in the microbiological assay of folate. Food Chemistry. 1992,43: 57-64.
67. Green JM, Merkel WK, Nichols BP. Characterization and sequence of Escherichia coli PabC, the gene encoding aminodeoxychorismate lyase, a pyridoxal phosphate-containing enzyme. J. Bacteriol. 1992,174: 5317-5323.
68. Gregory JF, Manley DB, Day BPF. Fluorometric determination of folacin in biological materials using high performance liquid chromatography. J. Nutr. 1984,114: 341-353.
69. Gregory JF. Chemical and nutritional aspects of folate research:analytical procedures,methods of folate synthesis,stability and bioavailability of dietary folates. Adv. Food Nutr. Res. 1989, 33: 1-101.
70. Guerineau F, Brooks L, Meadows J, Lucy A, Robinson C, Mullineaux P. Sulfonamide resistance gene for plant transformation. Plant Mol Biol. 1990, 15: 127-36.
71. Gutteridge S, Gatenby AA. Rubisco synthesis, assembly, mechanism and regulation. Plant Cell. 1995,7:809-819.
72. Hampele I C, D'Arcy A, Dale GE, Kostrewa D, Nielsen J, Oefher C, Page MGP, Schonfeld HJ, Stuber D, Then RL. Structure and function of the dihydropteroate synthase from Staphylococcus aureus. J. Mol. Biol. 1997, 268: 21-30.
73. Hanson AD, Gregory JF. Synthesis and turnover of folates in plants. Curr. Opin. Plant Biol. 2002, 5: 244-249.
74. Harborne JB: Twenty-five years of chemical ecology. Nat Prod Rep 2001, 18: 361-379.
75. Harris MJ, Juriloff DM. Mini-review: toward understanding mechanisms of genetic neural tube defects in mice. Teratology. 1999, 60: 292-305.
76. Hawkes JG, Villota R. Folates in food: reactivity, stability during processing and nutritional implications. Crit Rev Food Sci Nutr .1989,28: 439-538.
77. Hennig M, Dale GE, D'Arcy A, Danel F, Fischer S, Gray CP, Jolidon S, Muller F, Page MGP, Pattison P, Oefher C. The structure and function of the 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Haemophilus influenzae. J. Mol. Biol. 1999,287: 211-219.
78. Hong Q, Ian A, Shan X, Yun-Jung C, Takamasa T, Barry S. Folylpoly-g-glutamate synthetase: generation of isozymes and the role in one carbon metabolism and antifolate cytotoxicity. Advan. Enzyme Regul. 1999, 39: 263-273.
79. Home DW, Patterson D, Cook RJ. Effect of nitrous oxide inactivation of vitamin B_(12)-dependent methionine synthetase on the subcellular distribution of folate coenzymes in rat liver. Arch. Biochem. Biophys. 1989, 270: 729-733.
80. Home DW, Patterson D. Lactobacillus casei Microbiological Assay of Folic acid Derivatives in 96 Well Microtiter Plates, Clin. Chem. 1988, 34(1): 2357-2359.
81. Hughes JJS, John MS, Helene M. The effect of different cooking methods on folate retention in various foods that are amongst the major contributors to folate intake in the UK diet British Journal of Nutrition. 2002, 88: 681-688.
82. Hurt E, Pesold-Hurt B, Schatz G The amino-terminal region of an importated mitochondrial precursor polypeptide can direct cytoplasmic dihydrofolate reductase into the mitochondrial matrix. EMBO J. 1984, 3: 3149-3156.
83. Iwai K, Dceda M, Kobashi M. Intracellular distribution, purification, and properties of dihydrofolate synthetase from pea seedlings. Methods Enzymol. 1980,66: 581-585.
84. Jain AK, Nessler CL. Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants. Mol Breeding, 2000, 6: 73-78.
85. John S, Fabrice R, John F. Review Folic acid and folates: the feasibility for nutritional enhancement in plant foods. J. Sci. Food Agric. 2000, 80: 795-824.
86. Kellam P, Dallas W, Ballantine SP, Delves CJ. Functional cloning of the dihydropteroate synthase gene of Staphylococcus haemolyticus. FEMS Microbiol. Lett. 1995, 134: 165-169.
87. Kim JS, Shane B. Role of folylpolyglutamate synthetase in the metabolism and cytotoxicity of 5-deazacyclotetrahydro-folate, an antipurine drug. J. Biol. Chem. 1994,269: 9714-9720.
88. Kohashi M. Isolation of six unconjugated pteridines from soybean (Glycin max L. Tsurunoko) seeds. J Biochem (Tokyo), 1980, 87(6): 1581-586.
89. Konings EJ. A validated liquid chromatographic method for determining folates in vegetables, milk powder, liver, and flour. J. AOAC Int. 1999, 82: 119-127.
90. Landgren F, Israelsson B, Lindgren A, Hultberg B, Andersson A, Brattstrom L. Plasma homocysteine in acute myocardial infarction: homocysteine-lowering effect of folic acid. J. Intern. Med. 1995, 37:381-388.
91. Lazar G, Zhang H, Goodman HM. The origin of the bifunctional dihydrofolate reductase-thymidylate synthase isogenes of Arabidopsis thaliana. Plant J. 1993, 3: 657-68.
92. Lee SW, Lee HW, Chung HJ, Kim YA, Kim YJ, Hahn Y, Chung JH, Park YS. Identification of the genes encoding enzymes involved in the early biosynthetic pathway of pteridines in Synechocystis sp. PCC 6803. FEMS Microbiol. Lett. 1999, 176: 169-176.
93. Leichter J, Switzer VP, Landymore AF. Effect of cooking on folate content of vegetables. Nutrition Reports International. 1978,18: 475-479.
94. Leichter J. Folate content in the solid and liquid portions of canned vegetables. Can. Inst. Food Sci. Technol. J. 1980, 13: 33-34.
95. Li JS. The multiple vitamin status of Chinese pregnant women with anemia and nonanemia in the last trimester. Journal-of-Nutritional-Science-and-Vitaminology. 2004, 50: 87-92
96. Lopez P, Lacks SA. A bifunctional protein in the folate biosynthetic pathway of Streptococcus pneumoniae with dihydropterin aldolase and hydroxymethyldihydropterin pyrophosphokinase activities. J. Bacteriol. 1993, 175: 2214-2220.
97. Lorimer GH. The carboxylation and oxygenation of ribulose 1,5-bisphosphate: the primary events in photosynthesis and photorespiration. Annu. Rev. Plant Physiol. 1981, 32: 349-383.
98. Luo M, Orsi R, Patrucco E, Pancaldi S, Cella R. Multiple transcription start sites of the carrot dihydrofolate reductasethymidylate synthase gene, and sub-cellular localization of the bifunctional protein. Plant Mol. Biol. 1997, 33: 709-722.
99. Luo M, Piffanelli P, Rastelli L, Cella R. Molecular cloning and analysis of a cDNA coding for the bifunctional dihydrofolate reductase-thymidylate synthase of Daucus carota. Plant Mol. Biol. 1993,22: 427-35.
100. Madison, WI. investigation with reversed-phase liquid chromatography. Clin. Chem. 1996, 42: 1847-1854.
101. Marco P, Moroni A, Merello E, Francechis R, Andreussi L, Finnell RH, Barber RC, Cama A, Capra V. Folate pathway gene alterations in patients with neural tube defects. Am. J. Med. Genet. 2000, 95: 216-223.
102. Mauch F, Mauch-Mani B, Gaille C, Kull B, Haas D, Reimmann C. Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase. Plant J. 2001, 25:67-77.
103. McBurney MW, Whitmore GF. Isolation and biochemical characterization of folate deficient mutants of Chinese hamster cells. Cell. 1974, 2: 173-182.
104. McDonald D, Atkinson IJ, Cossins EA, Shane B. Isolation of dihydrofolate and folylpolyglutamate synthetase activities from Neurospora. Phytochemistry, 1995, 38: 327-333.
105. McGuire JJ, Bertino JR. Enzymatic synthesis and function of polyglutamates. Mol. Cell Biochem. 1981,38: 19-48.
106. McNeil SD, Nuccio ML, Ziemak MJ, Hanson AH. Enhanced synthesis of choline and glycine betaine in transgenic tobacco plants that overexpress phosphoethanolamine N-methyltransferase.Proc. Natl. Acad. Sci. USA, 2001, 98: 10001-10005.
107. Miller CF, Guadagni DG, Kon S. Vitamin retention in bean products: cooked, canned and instant bean powders. Journal of Food Science. 1973, 38: 493-495.
108. Milstien S, Jaffe H, Kowlessur D, Bonner TI. Purification and cloning of the GTP cyclohydrolase I feedback regulatory protein, GFRP. J. Biol. Chem. 1996, 271: 19743-19751.
109. Moloy AM, Scott JM. Microbiological Assay for Serum, Plasma and Red Cel Folate Using Cryopreserved, Microtiter Plate Method. Methodes in enzymology, 1997,281: 43-53.
110. Morris MS. Folate homocysteine and neurological function. Nutr. Clin. Care. 2002, 5: 124:132.
111. Muir SR, Collins GJ, Robinson S, Hughes SG, Bovy AG, Vos CH, Tunen AJ, Verhoeyen ME. Overexpression of petunia chalcone isomerase in tomato results in fruit containing dramatically increased levels of flavonols. Nat. Biotechnol. 2001,19,470-474.
112. Nar H, Huber R, Auerbach G, Fischer M, Ho'sl C, Ritz H, Bracher A, Meining W, Eberhardt S, Bacher A. Active site topology and reaction mechanism of GTP cyclohydrolase I. Proc. Natl. Acad. Sci. USA, 1995,92: 12120-12125.
113. Nardese V, Gutlich M. Brambilla A, Carbone ML. Biochem. Biophys. Res. Commun. 1996,218: 273-279.
114. Nelen, WL, Blom HJ, Steegers EA, Heijer M, Thomas CM, Eskes TK. Homocysteine and folate levels as risk factors for recurrent early pregnancy loss. Obstet. Gynecol. 2000, 95: 519-524.
115. Neuburger M, Rebeille F, Jourdain A, Nakamura S, Douce R. Mitochondria are a major site for folate and thymidylate synthesis in plants. J. Biol. Chem. 1996, 271: 9466-9472.
116. Nichols BP, Seibold AM, Doktor SZ. Para-aminoben-zoate synthesis from chorismate occurs in two steps. J. Biol. Chem. 1989,264: 8597-8601.
117. Okinaka O, Iwai K. The biosynthesis of folic acid compounds in plants. III. Distribution of the dihydropteroate synthesizing enzyme in plants. J. Vitaminol 1970, 16: 196-200.
118. Pedersen JC. Comparison of gamma glutamyl transferase hydrolase (conjugase EC3.4.22.12) and amylase treatment procedures in the microbiological assay for food folates. Brit. J. Nutrition, 1988,59:261-271.
119. Peng M, Sanchez-Fernandez R, Chen S, Rea PA. The Arabidopsis full-molecule ABC protein AtMRP4 has the characteristics of a folic acid transporter. Abstract 82 of the 12th International Workshop in Plant Membrane Biology. 2001, August 11-16.
120. Petersen BL, Andreasson E, Bak S, Agerbirk N, Halkier BA. Characterization of transgenic Arabidopsis thaliana with metabolically engineered high levels of p-hydroxybenzylglucosinolate. Planta, 2001,212: 612-618.
121. Pfeiffer CM, Gregory JF. Enzymatic deconjugation of erythrocyte polyglutamyl folates during preparation for folate assay:investigation with reversed-phase liquid chromatography. Clin. Chem. 1996,42: 1847-1854.
122. Pfeiffer CM, Rogers LM, Gregory JF. Determination of folate in cereal-grain food products using tri-enzyme extraction and combined affinity and reverse-phase liquid chromatography. J. Agric. Food Chem. 1997,45: 407-413.
123. Phillips DR, Wright AJA. Studies on the response of Lactobacillus casei to different folate monoglutamates. Br. J. Nutr. 1982,47: 183-189.
124. Phillips DR, Wright AJA. Studies on the response of Lactobacillus casei to folate vitamine in foods. Br. J. Nutr. 1983,49: 181-186.
125. Pietrzik K. Nutrients considered worthy of examination in processed foods, in Thermal Processing and Quality of Foods, Ed by Zeuthen et al. Elsevier Applied Science, London. 1984, pp825.
126. Prabhu V, Chatson KB, Lui H, Abrams GD, King J. Effects of sulfanilamide and methotrexate on ~(13)C fluxes through the glycine decarboxylase/serine hydroxymethyltransferase enzyme system in Arabidopsis. Plant Physiol. 1998,116: 137-144.
127. Put NM, Straayen HW, Trijbels FJ, Blom HJ. Folate, homocysteine and neural tube defects: an overview. Exp. Biol.Med. 2001,226: 243-270.
128. Quinlivan EP, Roje S, Basset G, Shachar-Hill Y, Gregory JF, Hanson A.D. The folate precursor p-aminobenzoate isreversibly converted to its glucose ester in the plant cytosol. J. Biol.Chem. 2003,278:20731-20737.
129. Rampersaud GC, Kauwell GP, Bailey LB. Folate: a key to optimizing health and reducing disease risk in the elderly. J.Clin. Nutr. 2003,22: 1-8.
130. Rebeille F, Douce R. Folate synthesis and compartmentation in higher plants, in Regulation of primary metabolic pathway in plants, Ed by Kruger NJ, Hill SA and Ratclife RG Kluwer Academic Publishers, Dordrecht, 1999, pp53-99.
131. Rebeille F, Macherel D, Mouillon JM, Garin J, Douce R. Folate biosynthesis in higher plants: purification and molecular cloning of a bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase 7,8-dihydropteroate synthase localized in mitochondria. EMBO J .1997, 16: 947-957.
132. Rijnboutt S, Jansen G, Posthuma G Hynes JB, Schornagel JH. Endocytosis of GPI-linked membrane folate receptor-a. J Cell Biol. 1996,132: 35-47.
133. Rimm EB, Willett WC, Hu FB, Sampson L, Colditz GA, Manson JE, Hennekens C, Stampfer MJ. Folate and vitamin B_(6) from diet and supplements in relation to risk of coronary heart disease among women. JAMA .1998,279: 359-364.
134. Robinson K, Arheart K, Refsum H, Brattstrom L, Boers G, Ueland P, Rubba P, Palma-Reis R, Meleasy R, Daly L, Witteman J, Graham I. Low circulating folate and vitamin B_(6) concentrations; risk factors for stroke, peripheral vascular disease, and coronary artery disease. Circulation. 1998, 97: 437-443.
135. Rocio DG, Eoin PQ, Sebastian MJK, Gilles JCB, Jesse FGIII Andrew DH. Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis. Proc. Natl. Acad. Sci. 2004,101: 13720-13725.
136. Roje S, Wang H, McNeil SD, Raymond RK, Appling DR, Shachar-Hill Y, Bohnert HJ, Hanson AD. Isolation, characterization, and functional expression of cDNAs encoding NADH-dependent methylenetetrahydrofolate reductase from higher plants. J. Biol. Chem. 1999, 274: 36089-36096.
137. Romer S, Fraser PD, Kiano JW, Shipton CA, Misawa N, Schuch W, Bramley PM. Elevation of the provitamin A content of transgenic tomato plants. Nature Biotechnol. 2000, 18: 666-669
138. Roos AJ, Cossins EA. Pteroylglutamate derivatives in Pisum sativum L. Biosynthesis of cotyledonary tetrahydropteroylglutamates during germination. Biochem. J. 1971, 125: 17-26.
139. Rosati C, Aquilani R, Dharmapuri S, Pallara P, Marusic C, Tavazza R, Bouvier F, Camara B, Giuliano G Metabolic engineering of β-carotene and lycopene content in tomato fruit. Plant J. 2000,24:413-419.
140. Rosenquist TH, Ratashak SA, Selhub J. Homocysteinine induces congenital defects of the heart and neural tube: effect of folic acid. Proc. Natl. Acad. Sci. USA. 1996,93: 15227-15232.
141. Ruggeri S, Vahteristo L, Aguzzi A, Finglas P. M, Carnovale E. Determination of folate vitamers in food and in Italian reference diet by high-performance liquid chromatography. Journal of Chromatography A, 1999, 855: 237-245.
142. Salcedo E, Cortese JF, Plowe CV, Sims PF, Hyde JEA. Mol. Biochem. A bifunctional dihydrofolate synthetase—folylpolyglutamate synthetase in Plasmodium falciparum identified by functional complementation in yeast and bacteria. 2001,112: 239-252.
143. Sauberlich HE, Kretsch MJ, Skala JH, Johnson HL, Taylor PC. Folate requirement and metabolism in nonpregnant women. Am J Clin Nutr. 1987,46: 1016-28.
144. Savidge B, Weiss JD, Wong YHH, Lassner MW, Mitsky TA, Showedmaker CK, Post-Beittenmiller D, Valentin HE. Isolation and characterization of homogentisate phytyltransferase genes from Synechocystis sp. PCC 6803 and Arabidopsis. Plant Physiol. 2002, 129:321-332
145. Schorah CJ, Devitt H, Lucock M, Dowell AC. The responsiveness of plasma homocysteine to small increases in dietary folic acid; a primary care study. Eur. J. Clin. Nutr. 1998, 52: 407-411.
146. Scott JM, Weir DG Folate/Vitamin B_(12) interrelationship. Essays in Biochemistry, 1994, pp63-72.
147. Scott JM, Weir DG Homocysteine and cardiovascular disease. Quarterly Journal of Medicine, 1996,89:561-563.
148. Scott JM. Folate and vitamin B_(12). Proc. Nutr. Soc. 1999, 58:441-448.
149. Selhub J, Jacques PF, Wilson PWF, Rush D, Rosenberg IH. Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA .1993, 270: 2693-2698.
150. Selhub J. Determination of tissue folate composition by affinity chromatography followed by high-pressure ion pair liquid chromatography. Anal. Biochem. 1989, 182:84-93.
151. Seyoum E, Selhub J. Combined affinity and ion pair column chromatographies for the analysis of food folate. J Nutr Biochem 1993,4:488-494.
152. Seyoum E, Selhub J. Combined affinity and ion pair column chromatographies for the analysis of food folate. J Nutr Biochem 1993,4:488-494.
153. Shane B. Folate Chemistry and Metabolism in Folate in Health and Disease, Ed by LB. Marcel Dekker, New York, 1995, pp1-22.
154. Shea TB, Lyons-Weiler J, Rogers E. Homocysteine, folate deprivation and Alzheimer neuropathology. J. Alzheimer Dis. 2002,4: 261-267.
155. Showedmaker CK, Sheehy JA, Daley M, Colburn S, Ke DY. Seed-specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects. Plant J. 1999, 20: 401-412.
156. Shin YS, Kim ES, Watson JE, Stokstad EL. Studies folic acid compounds in nature. IV. Folic acid compounds in soybeans and cow milk. Can. J. Biochem. 1975, 53: 338-343.
157. Shotkoski FA, Fallon AM. An amplied insect dihydrofolate reductase gene contains a single intron. Eur. J. Biochem. 1988, 27: 3391-3399.
158. Silvina FC, Jacob S, Peter WFW, Jeanne IR, Irwin HR, Paul FJ. Folic Acid Intake from Fortification in United States Exceeds Predictions. J. Nutr. 2002, 132: 2792-2798.
159. Stammers DK, Achari A, Somers DO, Bryant PK, Rosemond J, Scott DL, Champness JN. 2.0 AI X-ray structure of the ternary complex of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase from Escherichia coli with ATP and a substrate analogue. FEBS Lett. 1999,456: 49-53.
160. Stephane R, Helene C, Samuel J, Didier G, Yolande SK, Roland D, Fabrice R. Tetrahydrofolate biosynthesis in plants: Molecular and functional characterization of dihydrofolate synthetase and three isoforms of folylpolyglutamate synthetase in Arabidopsis thaliana. Proc. Natl. Acad. Sci.USA, 2001, 98: 15360-15365.
161. Subar AF, Block G, James LD. Folate intake and food sources in the U.S. population. Am. J. Clin. Nutr. 1989,50:508-516.
162. Sungsoo C, Guy J, Won OS. Folate Content of Foods: Comparison Between DatabasesCompiled Before and After New FDA Fortification Requirements. Journal of Food Composition and Analysis, 2002, 15:293-307.
163. Suzuki JY, Bollivar DW, Bauer CE. Genetic analysis of chlorophyll biosynthesis. Ann. Rev. Genet. 1997,31:61-89.
164. Swafford JR, Beverley SM, Kan CC, Caulfield JP. Distribution of dihydrofolate reductase-thymidylate synthetase polypeptide in methotrexate resistant and wild type Leishmania major: evidence for cytoplasmic distribution despite a hydrophilic leader sequence, in: Proceedings of the XIlth congress for electron microscopy, Ed by Peachey LD and Williams DB. San Francisco Press, San Francisco, 1990, pp610-611.
165. Tahzeeba H, twin R, Jacob S, Ganesh K, Roger B, Karel S. Enhancement of folates in plants through metabolic engineering. Proc. Natl. Acad. Sci. 2004 , 101: 5158-5163.
166. Talarico TL, Ray PH, Dev IK, Merill B, Dallas WS. Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase. J. Bacteriol. 1992, 174: 5971-5977.
167. Tamura T. Determination of food folate. Journal of Nutritional Biochemistry. 1998, 9:285-293.
168. Titus SA, Moran RG Retrovirally mediated complementation of the glyB phenotype. Cloning of a human gene encoding the carrier for entry of folates into mitochondria. J. Biol. Chem. 2000, 275:36811-36817.
169. Triglia T, Cowman AF. Primary structure and expression of the dihydropteroate synthase gene of Plasmodium falciparum. Proc. Natl. Acad. Sci. USA, 1994, 91: 7149-7153.
170. Vahteristo LT, Lehikoinen K, Ollilainen V, Varo P. Application of a HPLC assay for the determination of folate derivates in some vegetables, fruit and berries consumed in Finland. Food Chem. 1997, 59: 589-97.
171. Verho P, Stampfer MJ, Buring JE, Gaziano JM, Allen RH, Stabler SP, Reynolds RS, Kok FJ, Hennekens CH, Willett WC. Homocysteine metabolism and risk of myocardial infarction: relation with vitamins B_(6), B_(12), and folate. Am. J. Epidemiol. 1996, 143: 845-859.
172. Verhoef P, Stampfer MJ, Rimm EB. Folate and coronary heart disease. Curr. Opin. Lipidol. 1998, 9: 17-22.
173. Verpoorte R, Alfermann AW(Eds). Metabolic Engineering of Plant. Secondary Metabolism. Dordrecht, Kluwer Academic Publishers, 2000.
174. Vikham P, Heien L, John K. Arabidopsis dihydropteroate synthase: general properyies and inhibition by reaction product and sulfonamides. Phytochemistry. 1997,45: 23-27.
175. Vikham P, Heien L, John K. Arabidopsis dihydropteroate synthase: general properyies and inhibition by reaction product and sulfonamides. Phytochemistry, 1997, 5: 23-27.
176. Volpe F, Ballantine SP, Delves CJ. The multifunctional folic acid synthesis fas gene of Pneumocystis carinii encodes dihydroneopterin aldolase, hydroxymethyldihydropterin pyrophosphokinase and dihydropteroate synthase. Eur. J. Biochem. 1993,216: 449-458.
177. Wald NJ, Watt HC, Law MR, Weir DG, McPartlin J, Scott JM. Homocysteine and ischaemic heart disease: results of a prospective study with implications on prevention. Arch of Internal Medicine 1998,158:862-867.
178. Wang M, Ratnam S, Freisheim JH. Cloning, nucleotide sequence and expression of the biftinctional dihydrofolate reductase-thymidylate synthase from Glycine max. Biochim. Biophys. Acta. 1995, 1261:325-36.
179. Waters AH, Mollin DL. Observations on the metabolism of folic acid in pernicious anaemia. Brit. J. Haematol. 1963, 9: 319-327.
180. Wei MM, Gregory JF. Organic acids in selected foods inhibit intestinal brush border pteroylpolyglutamate hydrolase in vitro: potential mechanism affecting the bioavailability of dietary polyglutamyl folate. Journal of Agricultural and Food Chemistry .1998,46: 211-219.
181. Wickramasinghe SN, Fida S. Bone marrow cells from vitamin B_(12)- and folate-deficient patients misincorporate uracil into DNA. Blood 1994, 83: 1656-1661.
182. Woodside JV, Yarnell JWG, McMaster D, Young IS, Harmon DL, Mccrum EE, Patterson CC, Gey KF, Whitehead AS, Evans A. Effect of B-group vitamins and antioxidant vitamins on hyperhomocysteinemia: a double-blind, randomized, factorial-design,controlled trial. Am. J. Clin. Nutr. 1998, 67: 858-866.
183. Wu K, Cossins EA, King J. Folate metabolism in Datura innoxia. In vivo and in vitro folylpolyglutamate synthesis in wild-type and methotrexate-resistant cells. Plant Physiol. 1994,
