摘要
酰基辅酶A:胆固醇酰基转移酶(ACAT)是细胞内唯一催化游离胆固醇和脂肪酸合成胆固醇酯的酶,在细胞内胆固醇代谢平衡中起非常重要的作用。目前,在哺乳动物细胞中发现两种基因编码ACAT(ACAT1和ACAT2)。ACAT1几乎在各种组织细胞中均有表达,与细胞胆固醇代谢平衡直接相关;而在肝肠细胞中特异表达的ACAT2则与外源胆固醇的吸收和脂蛋白装配等密切相关。人类动脉粥样硬化、阿尔海默氏病、胆囊结石等一些重要病变,都与ACAT密切相关。因此,ACAT已成为分子筛药的重要靶蛋白。我们实验室与美国Chang TY教授实验室的合作研究,已首先报道了两种人ACAT基因的基因组结构和ACAT1基因的表达调控。在这些基础上,本论文进行两部分工作,第一部分重点围绕人ACAT2基因表达的可变性剪接和组织特异性进行研究,第二小部分是进行猴ACAT1 cDNA 5′-UTR的克隆分析。
首先,进行可变性剪接成熟的人ACAT2 mRNAs及其编码的异构体酶活性的分析研究。人ACAT2基因长约18 kb,包含15个外显子,所有的外显子/内含子连接区序列均为经典的GT/AG序列。通过RT-PCR、克隆和DNA测序分析,获得三种人ACAT2 mRNAs。序列比较表明,它们是从同一pre-mRNA通过可变性剪接产生,外显子的缺失并未导致阅读框的改变;它们编码三种人ACAT2产物,即ACAT2a,包含所有的15个外显子,编码522个氨基酸;ACAT2b,包含除外显子4以外的14个外显子,编码502个氨基酸;ACAT2c,包含外显子1-3、6-7、11-15,编码379个氨基酸;其中ACAT2a与文献报道完全一致,ACAT2b与ACAT2c为新发现的2个ACAT2序列。利用克隆的三种大小不同的人ACAT2 ORF cDNAs构建相应的表达质粒,分别转染ACAT缺陷的AC29细胞进行表达研究,结果显示可表达出相应不同大小的ACAT2蛋白产物;同时在人肝肠细胞株中也检测到了同样不同大小的内源表达ACAT2蛋白产物。这些表明,可表达的内源性ACAT2b和ACAT2c蛋白可能是两种ACAT2异构体,而且它们的氨基
Acyl-CoA: cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the formation of cholesterol esters from long-chain fatty acyl coenzyme A and cholesterol. ACAT plays an important role in cellular cholesterol homeostasis. So far, in mammals, two ACAT genes have been identified. The ACAT1 that is found in almost all of the cells and tissues examined is believed to be crucial for maintaining cholesterol homeostasis while ACAT2 that is selectively expressed in liver and small intestine may be the major enzyme that involved in the dietary cholesterol absorption and assembly of apoB-containing lipoprotein. ACATs also play important roles in some serious human diseases including atherosclerosis, Alzheimer’s disease (AD) and gallstone. For these reasons, ACAT has been considered as one of major pharmaceutical targets for developing CE-lowering, anti-atherosclerosis and/or anti-AD drugs. In collaboration with professor Chang TY′s laboratory (Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA), our laboratory (State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China) has first reported the two human ACAT genomic DNAs, and investigated the expressional regulation of human ACAT1 gene. Based on the previous study, this work is mainly focused on the alternative splicing and tissue-specificity of human ACAT2 gene expression, and cloning and analysis of monkey ACAT1 cDNA 5′-UTR.
Firstly, we studied the human ACAT2 mRNA variants from alternative splicing
引文
Simons K, Ikonen E. Functional rafts in cell membranes. Nature (1997) 387: 569-572
Anderson RGW. The caveolae membrane system. Annu. Rev. Biochem. (1998) 67: 199-225
Buhman KF, Accad M, Farese RV. Mammalian acyl-CoA:cholesterol acyltransferases.
Biochim Biophys Acta. (2000) Dec 15; 1529(1-3): 142-54. Review
Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science (1986) 232: 34-47
Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell (1997) 89: 331-340
Chang TY, Chang CCY, Chen D. Acyl-Coenzyme A: cholesterol acyltransferase. Ann. Rev. Biochem. (1996) 66: 613-638
Mukherjee S, Kunitake G, Alfin-Slater RB. The esterification of cholesterol with palmitic acid by rat liver homogenates. J Biol Chem. (1958) Jan; 230(1): 91-96.
Chang CCY, Huh HY, Cadigan KM, Chang TY. Molecular cloning and functional expression of human acyl-coenzyme A:cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells. J. Biol. Chem. (1993) 268: 20747-20755
Yang H, Bard M, Bruner DA, Gleeson A, Deckelbaum RJ, Aljinovic G, Pohl TM, Rothstein R, Sturley SL. Sterol esterification in yeast: a two-gene process. Science (1996) 272(5266): 1353-1356
Meiner VL, Cases S, Myers HM, Sande ER, Bellosta S, Schambelan M, Pitas PE, McGuire J, Herz J, Farese RV. Disruption of the acyl-CoA:cholesterol acyltransferase gene in mice: evidence suggesting multiple cholesterol esterification enzymes in mammals. Proc Natl Acad Sci U S A. (1996) 93(24): 14041-14046
Cases S, Novak S, Zheng Y, Myers HM, Lear SR, Sande, E, et al. ACAT-2, A second mammalian acyl CoA: chelesterol acyltransferase: its cloning, expression, and characterization. J. Biol. Chem. (1998) 273: 26755-26764.
Anderson RA, Joyce C, Davis M, Reagan JW, Clark M, Sheiness GS, Rudel LL. Identification of a form of acyl-CoA: cholesterol acyltransferase specific to liver and intestine in nonhuman primates. J. Biol. Chem. (1998) 273: 26747-26754
Oelkers P, Behari A, Cromley D, Billheimer JT, Sturley SL. Characterization of two human genes encoding acyl coenzyme A-cholesterol acyltransferase related enzymes. J. Biol. Chem. (1998) 273: 26765-26771
Li BL, Li XL, Duan ZJ, Lee O, Lin s, Ma ZM, Chang CC, Yang XY, Park JP, Mohandas TK, Noll W, Chan L, Chang TY. Human acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) gene organization and evidence that the .3-kilobase ACAT-1 mRNA is produced from two different chromosomes. J Biol Chem. (1999) 274(16): 11060-11071
Song BL, Qi W, Yang XY, Chang CC, Zhu JQ, Chang TY, Li BL. Organization of human ACAT-2 gene and its cell type-specific promoter activity. Biochem Biophys Res Commun. (2001) 282(2): 580-588
Rudel LL, LL RG, Cockman TL. Acyl coenzyme A: cholesterol acyltransferase types 1 and 2: structure and function in atherosclerosis. Curr Opin Lipidol. (2001) Apr;12(2): 121-127. Review
Turley SD and DietschyJM. The metabolism and excretion of cholesterol by the liver[A]. Arias IM, Jakoby WB, Popper H, et al. The liver: biology and pathology[M]. New York: Raven Press, (1998) 617-641
Chang TY, Chang CC, Lu X, Lin S. Catalysis of ACAT may be completed within the plane of the membrane: a working hypothesis. J. Lipid Res. (2002) 42: 1933-1938
Carr TP, Hamilton RL, Rudel LL. ACAT inhibitors decrease secretion of cholesteryl esters and apolipoprotein B by perfused livers of African green monkeys. J.Lipid.Res. (1995) 36(1): 25-36
Tanaka M, Jingami H, Otani H, Cho M, Ueda Y, Arai H, Nagano Y, Doi T, Yokode M, Kita T. Regulation of apolipoprotein B production and secretion in response to the change of intracellular cholesteryl ester contents in rabbit hepatocytes. J Biol Chem. (1993) 268(17): 12713-12718
Wu X, Sakata N, Lui E, Ginsberg HN. Evidence for a lack of regulation of the assembly and secretion of apolipoprotein B-containing lipoprotein from HepG2 cells by cholesteryl ester. J Biol Chem. (1994) 269(16): 12375-12382
Yang JB, Duan ZJ, Yao W, Lee O, Yang L, Yang XY, Sun X, Chang CCY, Chang TY, Li BL. Synergistic transcriptional activation of human ACAT-1 gene by IFN-γ and ATRA in THP-1 cells. J. Biol. Chem. (2001) 276: 20989-20998
Accad M, Smith SJ, Newland DL, Sasan DA, King LE, Linton MF, Fazio S, Farese RV. Massive xanthomatosis and altered composition of atherosclerotic lesions in hyperlipidemic mice lacking acyl CoA:cholesterol acyltransferase 1. J Clin Invest. (2000) 105(6): 711-719
Yagyu H, Kitamine T, Osuga J, Tozawa R, Chen Z, Kaji Y, Oka T, Perrey S, Tamura Y, Ohashi K, Okazaki H, Yahagi N, Shionoiri F, Iizuka Y, Harada K, Shimano H,
Yamashita H, Gotoda T, Yamada N, Ishibashi S. Absence of ACAT-1 attenuates atherosclerosis but causes dry eye and cutaneous xanthomatosis in mice with congenital hyperlipidemia. J Biol Chem. (2000) 275(28): 21324-21330
Fazio S, Major AS, Swift LL, Gleaves LA, Accad M, Linton MF, Farese RV Jr. Increased atherosclerosis in LDL receptor-null mice lacking ACAT1 in macrophages. J Clin Invest. (2001) 107(2): 163-171
Buhman KK, Accad M, Novak S, Choi RS, Wong JS, Hamilton RL, Turley S, Farese RV Jr. Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice. Nat Med. (2000) 6(12): 1341-1347
Scott HD, Laake K. Statins for the prevention of Alzheimer's disease (Cochrane Review). The Cochrane Library, Issue 4, Chichester, UK: John Wiley & Sons, Ltd (2003)
Puglielli L, Konopka G, Pack-Chung, E, Ingano LAM, Berezovska, O, Hyman BT, Chang TY, Tanzi RE, Kovacs DM. Acyl-coenzyme A: cholesterol
acyltransferase modulates the generation of the amyloid β-peptide. Nature Cell Biololy. (2001) 3: 905-912
Hutter-Paier B, Huttunen HJ, Puglielli L, Eckman CB, Kim DY, Hofmeister A, Moir RD, Domnitz SB, Frosch MP, Windisch M, Kovacs DM. The ACAT2 Inhibitors CP-113,818 Markedly Reduces Amyloid Pathology in a Mouse Model of Alzheimer′s Disease. Neuron (2004) 44: 227-238
Pape ME, Schultz PA, Rea TJ, DeMattos RB, Kieft K, Bisgaier CL, Newton RS, Krause BR. Tissue specific changes in acyl-CoA: cholesterol acyltransferase (ACAT) mRNA levels in rabbits. J Lipid Res. (1995) 36(4): 823-838
Wang H, Germain SJ, Benfield PP, Gillies PJ. Gene expression of acyl-coenzyme-A:cholesterol-acyltransferase is upregulated in human monocytes during differentiation and foam cell formation. Arterioscler. Thromb. Vasc. Biol. (1996) 16: 809-814
Miyazaki A, Sakashita N, Lee O, Takahashi K, Horiuchi S, Hakamata H, Morganelli PM, Chang CCY, Chang TY. Expression of ACAT-1 protein in human atherosclerotic lesions and cultured human monocytes-macrophages. Arterio. Throm. Vas. Biol. (1998) 18: 1568-1574
Cheng W, Kvilekval KV, Abumrad NA. Dexamethasone enhances accumulation of cholesteryl esters by human macrophages. Am. J. Physiol. (1995) 269: E642-E648
Panousis CG, Zuckerman SH. Regulation of cholesterol distribution in macrophage-derived foam cells by interferon-gamma. J. Lipid Res. (2000) 41: 75-83
Yang L, Yang JB, Chen J, Yu JY, Zhou P, Lei L, Wang ZZ, Cy Chang C, Yang XY, Chang TY, Li BL. Enhancement of human ACAT1 gene expression to promote the macrophage-derived foam cell formation by dexamethasone. Cell Res. (2004) Aug;14(4): 315-323
Chang CCY, Sakashita N, Ornvold K, Lee O, Chang ET, Dong R, Lin S, Lee CY, Strom SC, Kashyap R, Fung JJ, Farese RV Jr, Patoiseau JF, Delhon A, and Chang TY. Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine. J. Biol. Chem. (2000) 275: 28083-28092
Li XL, Duan ZJ, Chang TY, and Li BL. Experimental evidences for trans-splicing in the 4.3-kilobase human ACAT-1 mRNA proceeding. Presentation at The 2000-4th A-IMBN Conference, (2000)
Yang L, Lee O, Chen J, Chen J, Chang CCY, Zhou P, Wang ZZ et al. Human acyl-coenzyme A:cholesterol acyltransferase 1 (acat1) sequences located in two different chromosomes (7 and 1) are required to produce a novel ACAT1 isoenzyme with additional sequence at the N terminus. J Biol Chem. (2004) 279: 46253-46262
Lin S, Cheng D, Liu MS, Chen J, Chang TY. Human acyl-CoA:cholesterol acyltransferase-1 in the endoplasmic reticulum contains seven transmembrane domains. J. Biol. Chem. (1999) 274: 23276-23285
Lin S, Lu X, Chang CCY, Chang TY. Human acyl-coenzyme a:cholesterol acyltransferase expressed in chinese hamster ovary cells: membrane topology and active site location. Mol. Biol. Cell. (2003) 14: 2447-2460
Joyce CW, Shelness GS, Davis MA, Lee RG, Skinner K, Anderson RA, Rudel LL. ACAT1 and ACAT2 membrane topology segregates a serine residue essential for activity to opposite sides of the endoplasmic reticulum membrane. Mol. Biol. Cell (2000) 11: 3675-3687
Yu CJ, Chen J, Lin S, Liu J, Chang CCY, Chang TY. Human acyl-CoA:cholesterol acyltransferase-1 is a homotetrameric enzyme in intact cells and in vitro. J. Biol. Chem. (1999) 274: 36139-36145
Yu CJ, Zhang Y, Lu XH, Chen J, Chang CCY, Chang TY. Role of the N-terminal hydrophilic domain of acyl-coenzyme A:cholesterol acyltransferase 1 on the enzyme's quaternary structure and catalytic efficiency. Biochemistry (2002) 41: 3762-3769
Cheng D, Chang CC, Qu X, Chang TY. Activation of acyl-coenzyme A:cholesterol acyltransferase by cholesterol or by oxysterol in a cell-free system. J. Biol. Chem. (1995) 270: 685-695
Chang CCY, Lee CY, Chang ET, Cruz JC, Levesque MC, Chang TY. Recombinant acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesicles in a highly cooperative manner. J. Biol. Chem. (1998) 273: 35132-35141
Lu X, Lin S, Chang CC, Chang TY. Mutant Acyl-coenzyme A:Cholesterol Acyltransferase 1 Devoid of Cysteine Residues Remains Catalytically Active. J. Biol. Chem. (2002) 277: 711-718
Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell (1997) 89: 331-340
Brown MS, Goldstein JL. A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood. Proc. Natl. Acad. Sci. USA (1999) 96: 11041-11048
Repa JJ, Mangelsdorf. The roles of orphan nuclear receptors in the regulation of cholesterol homeostasis. Annu. Rev. Cell Dve. Biol. (2000) 16: 459-481
Matsuda H, Hakamata H, Kawasaki T, Sakashita N, Miyazaki A, Takahashi K, Shichiri M. Horiuchi,S. Molecular cloning, functional expression and tissue distribution of rat acyl-coenzyme A:cholesterol acyltransferase. Biochim. Biophys. Acta (1998) 1391 (2): 193-203
Uelmen PJ, Oka K, Sullivan M, Chang CC, Chang TY, Chan L. Tissue-specific expression and cholesterol regulation of acylcoenzyme A:cholesterol acyltransferase (ACAT) in mice. Molecular cloning of mouse ACAT cDNA, chromosomal localization, and regulation of ACAT in vivo and in vitro. J. Biol. Chem. (1995) 270 (44): 26192-26201
Green S, Steinberg D, Quehenberger O. Cloning and expression in Xenopus oocytes of a mouse homologue of the human acylcoenzyme A: cholesterol
acyltransferase and its potential role in metabolism of oxidized LDL. Biochem. Biophys. Res. Commun. (1996) 218 (3): 924-929
Maniatis T, Tasic B. Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature (2002) 418: 236-243
Graveley BR. Alternative splicing: increasing diversity in the proteomic world. Trends in Genetics. (2002) 17: 100-107
Modrek B, Lee C. A genomic view of alternative splicing. Nature genetics. (2002) 30: 10-19
Li BL, Li XL, Duan ZJ, Lee O, Lin s, Ma ZM, Chang CC, Yang XY, Park JP, Mohandas TK, Noll W, Chan L, Chang TY. Human acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) gene organization and evidence that the 4.3-kilobase ACAT-1 mRNA is produced from two different chromosomes. J Biol Chem. (1999) 274(16): 11060-11071
Chang CC, Chen J, Thomas MA, Cheng D, Del Priore VA, Newton RS, Pape ME, Chang TY. Regulation and immunolocalization of acyl-coenzyme A: cholesterol acyltransferase in mammalian cells as studied with specific antibodies. J Biol Chem. (1995) 270: 29532-29540
Liu J, Streiff R, Zhang YL, Vestal RE, Spence MJ, Briggs MR. Novel mechanism of transcriptional activation of hepatic LDL receptor by oncostatin M. J Lipid Res. (1997) 38: 2035-2048
Yang JB, Duan ZJ, Yao W, Lee O, Yang L, Yang XY, Sun X, Chang CCY, Chang TY, Li BL. Synergistic Transcriptional Activation of Human ACAT-1 Gene by IFN-γ and ATRA in THP-1 cells. J. Biol. Chem. (2001) 276: 20989-20998
Oosta GM, Mathewson NS, Catravas GN. Optimization of Folin-Ciocalteu Reagent concertration in an automated Lowry protein assay. Anal Biochem. (1978) 89: 31-34
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (1970) 227: 680-685
Song BL, Qi W, Yang XY, Chang CCY, Zhu JQ, Chang TY, Li BL. Organization of human ACAT-2 gene and its cell-type-specific promoter activity. Biochem Biophys Res Commun. (2001) 282: 580-588
Katsuren K, Tamura T, Arashiro R, Takata K, Matsuura T, Niikawa N, Ohta T. Structure of the human acyl-CoA:cholesterol acyltransferase-2 (ACAT-2) gene and its relation to dyslipidemia. Biochim Biophys Acta (2001) 1531: 230-240
Oelkers P, Behari A, Cromley D, Billheimer JT, Sturley SL. Characterization of two human genes encoding acyl coenzyme A:cholesterol acyltransferase related enzymes. J Biol Chem. (1998) 273: 26765-26771
Chang CCY, Sakashita N, Ornvold K, Lee O, Chang ET, Dong R, Lin S. Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine. J Biol Chem. (2000) 275: 28083-28092
Parini P, Davis M, Lada AT, Erickson SK, Wright TL, Gustafsson U, Sahlin S, Einarsson C, Eriksson M, Angelin B, Tomoda H, Omura S, Willingham MC, Rudel LL. ACAT2 Is Localized to Hepatocytes and Is the Major Cholesterol-Esterifying Enzyme in Human Liver. Circulation (2004) 110: 2017-2023
Graveley BR. Alternative splicing: increasing diversity in the proteomic world. Trends in Genetics (2002) 17: 100-107
Modrek B, Lee C. A genomic view of alternative splicing. Nature genetics (2002) 30: 10-19
Bishop JE, Hajra AK. A method for the chemical synthesis of 14C-labeled fatty acyl coenzyme A’s of high specific activity. Anal Biochem. (1980) 106: 344-350
Chang CCY, Chen J, Thomas MA, Cheng D, Priore VAD, Newton RS, Pape ME, Chang TY. Regulation and immunolocalization of acyl-coenzyme A:cholesterol acyltransferase in mammalian cells as studied with specific antibodies. J. Biol. Chem. (1995) 270: 29532-29540
Chang CCY, Chang TY. Cycloheximide sensitivity in regulation of acyl coenzyme A:cholesterol acyltransferase activity in Chinese hamster ovary cells. 2. Effect of sterol endogenously synthesized. Biochemistry (1986) 25(7): 1700-1706
Cadigan KM, Heider JG, Chang TY. Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A:cholesterol acyltransferase activity. J Biol Chem. (1988) 263(1): 274-282
Torres J, Pulido R. The tumor suppressor PTEN is phosphorylated by the protein kinase CK2 at its C terminus. Implications for PTEN stability to proteasome-mediated degradation. J Biol Chem. (2001) 276: 993-998
Ahmed K, Davis AT, Wang H, Faust RA, Yu S, Tawfic S. Sifnificance of protein kinase CK2 nuclear signaling in neoplasia. J. Cellular Bioch. (2000) 35: 130-135. Pinna LA. Protein kinase CK2. Int. J. Bioche. Cell Biol. (1997) 29: 551-554
Hernandez ML, Martinez MJ, Lopez de Heredia M, Ochoa B. Protein phosphatase 1 and 2A inhibitors activate acyl-CoA:cholesterol acyltransferase and cholesterol ester formation in isolated rat hepatocytes. Biochim Biophys Acta. (1997) 1349(3): 233-241
Kusuhara H, Shimada O, Inui J. Effect of 25-hydroxycholesterol on cholesteryl ester formation in Caco-2 cells. Lipids (1992) 27(6): 478-480
Guo ZY, Lin S, Heinen JA, Chang CC, Chang TY. The active site His460 of human acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1) resides in a hitherto undisclosed transmembrane domain. J Biol Chem. (2005) 280: 37814-37826
Lin S, Lu X, Chang CCY, Chang TY. Human acyl-coenzyme A:cholesterol acyltransferase expressed in chinese hamster ovary cells: membrane topology and active site location. Mol Biol Cell (2003) 14: 2447-2460
Lin S, Cheng D, Liu MS, Chen J, Chang TY. Human acyl-CoA:cholesterol acyltransferase-1 in the endoplasmic reticulum contains seven transmembrane domains. J Biol Chem. (1999) 274: 23276-23285
Joyce CW, Shelness GS, Davis MA, Lee RG, Skinner K, Anderson RA, Rudel LL. ACAT1 and ACAT2 membrane topology segregates a serine residue essential for activity to opposite sides of the endoplasmic reticulum membrane. Mol Biol Cell (2000) 11: 3675-3687
Song BL, Qi W, Yang XY, Chang CCY, Zhu JQ, Chang TY, Li BL. Organization of human ACAT-2 gene and its cell-type-specific promoter activity. Biochem Biophys Res Commun. (2001) 282: 580-588
Azizkhan JC, Jensen DE, Pierce AJ, Wade M. Transcription from TATA-less promoters: dihydrofolate reductase as a model. Critical Reviews in Eukaryotic Gene Expression. (1993) 3(4): 229-254
Reynolds GA, Basu SK, Osborne TF, Chin DJ, Gil G, Brown MS, Goldstein JL, Luskey KL. HMG CoA reductase: a negatively regulated gene with unusual promoter and 5' untranslated region. Cell (1984) 38: 275
Valerio D, Duyvesteyn MG, Dekker BM, Weeda G, Berkvens TM, et al. Adenosine deaminase: Characterization and expression of a gene with a remarkable promoter. EMBO J. (1985) 4: 437
Hahn SL, Hahn M, Hayward WS. Structural organization of upstream exons and distribution of transcription start sites in the chicken c-myb promoter. Mol. Cell Biol. (1989) 9: 837
Blasband AJ, Rogers KT, Chen XR, Azizkhan JC, Lee DC. Characterization of the rat transforming factor alpha gene and identification of promoter sequences. Mol. Cell Biol. (1990) 10: 2111
Shibata F, Baird A, Florkiewicz RZ. Functional characterization of the human basic fibrablast growth factor gene promoter. Growth Factors (1991) 4: 277
Van-Dijk MA, Holthuizen PE, Sussenbach JS. Elements required for activation of the major promoter of the human insulin-like growth factor II gene. Mol. Cell Endocrinal. (1992) 88: 175
Ishii S, Merlino GT, Pastan I. Promoter region of the human Harvey ras protooncogene: similarity to the EGF receptor protooncogene promoter. Science (1985) 230: 1378
Sehgal A, Patil N, Chao M. A constitutive promoter directs expression of the nerve growth factor receptor gene. Mol. Cell Biol. (1988) 8: 3160
Chavrier P, Janssen-Timmen U, Mattei MG, Zerial M, Bravo R, Charnay P. Structure, chromosome location and expression of the mouse zinc finger gene Krox-20: multiple gene products and coregulation with the proto-oncogene c-fos. Mol. Cell (1989) 9: 787
Rim M, Qureshi SA, Guis D, Nho J, Sukhatme VP, Foster DA. Evidence that activation of the Egr-1 promoter by v-Raf involves serum response elements. Oncogene (1992) 7: 2065
Chang CCY, Sakashita N, Ornvold K, Lee O, Chang ET, Dong R, Lin S. Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine. J Biol Chem. (2000) 275: 28083-28092
Song BL, Qi W, Wang CH, Yang JB, Yang XY, Lin ZX, Li BL. Preparation of an anti-Cdx-2 antibody for analysis of different species Cdx-2 binding to acat2 promoter. Acta Biochim. Biophys. Sin. (2003) 35: 6-12
Yang JB, Duan ZJ, Yao W, Lee O, Yang L, Yang XY, Sun X, Chang CCY, Chang TY, Li BL. Synergistic Transcriptional Activation of Human ACAT-1 Gene by IFN-γ and ATRA in THP-1 cells. J. Biol. Chem. (2001) 276: 20989-20998
Liu J, Streiff R, Zhang YL, Vestal RE, Spence MJ, Briggs MR. Novel mechanism of transcriptional activation of hepatic LDL receptor by oncostatin M. J Lipid Res. (1997) 38: 2035-2048
Andrews NC, Faller DV. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. (1991) 19: 2499
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. (1976) 72:248-254
Mendel DB, Crabtree GR. HNF-1, a member of a novel class of dimerizing homeodomain protein. J. Biol. Chem. (1991) 266: 677-680
Chang TY, Chang CC, Lin S, Yu C, Li BL, Miyazaki A. Roles of acyl-coenzyme A:cholesterol acyltransferase-1 and –2. Curr Opin Lipidol. (2001) 12: 289-296
Chang TY, Chang CCY, Lu X, Lin S. Catalysis of ACAT may be completed within the plane of the membrane: a working hypothesis. J Lipid Res. (2001) Dec;42(12): 1933-1938. Review
Suh E, Chen L, Taylor J, Traber PG. A homeodomain protein related to caudal regulates intestine-specific gene transcription. Mol Cell Biol. (1994) Nov;14(11): 7340-7351
Lambert M, Colnot S, Suh E, L'Horset F, Blin C, Calliot M E, Raymondjean M et al. Cis-Acting elements and transcription factors involved in the intestinal specific expression of the rat calbindin-D9K gene: binding of the intestine-specific transcription factor Cdx-2 to the TATA box. Eur J Biochem. (1996) 236(3): 778-788
Drummond FJ, Sowden J, Morrison K, Edwards YH. The caudal-type homeobox protein Cdx-2 binds to the colon promoter of the carbonic anhydrase 1 gene. Eur. J. Biochem. (1996) 236(2): 670-681
Boudreau F, Zhu Y, Traber PG. Sucrase-isomaltase gene transcription requires the hepatocyte nuclear factor-1 (HNF-1) regulatory element and is regulated by the ratio of HNF-1 alpha to HNF-1 beta. J. Biol. Chem. (2001) 276: 32122-32128
Wu GD, Chen L, Forslund K, Traber PG. Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) and HNF-1 beta regulate transcription via two elements in an intestine-specific promoter. J Biol Chem. (1994) Jun 24; 269(25):17080-17085
Mitchelmore C, Troelsen JT, Spodsberg N, Sjostrom H, Noren O. Interaction between the homeodomain proteins Cdx-2 and HNF-1a mediates expression of the lactase-phlorizin hydrolase gene. Biochem. J. (2000) 346: 529-535
Sakaguchi T, Gu X, Golden HM, Suh E, Rhoads DB, Reinecker HC. Cloning of the human claudin-2 5’-flanking region revealed a TATA-less promoter with conserved binding sites in mouse and human for Caudal-Related Homeodomain Proteins and Hepatocyte Nuclear Factor-1a. J. Biol. Chem., epub. (2002) Lorentz O, Duluc I, Arcangelis AD, Simon-Assmann P, Kedinger M, Freund JN. J. Cell Biol. (1997) 139: 1553-1565
Shih DQ, Bussen M, Sehayek E, Ananthanarayanan M, Shneider BL, Suchy FJ, Shefer S, Bollileni JS, Gonzalez FJ, Breslow JL and Stoffel M. Hepatocyte nuclear factor-1alpha is an essential regulator of bile acid and plasma cholesterol metabolism. Nat Genet. (2001) 27(4): 375-382
Chang CCY, Sakashita N, Ornvold K, Lee O, Chang ET, Dong R, Lin S, Lee CY, Strom SC, Kashyap R, Fung JJ, Farese RV Jr, Patoiseau JF, Delhon A, Chang TY. Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine. J Biol Chem. (2000) 275(36): 28083-28092
Bennett Clark S, Tercyak AM. Reduced cholesterol transmucosal transport in rats inhibited mucosal acyl CoA: cholesterol acytransferase and normal pancreatic function. J. Lipid Res. (1984) 25: 148-159
Traber PG, Silberg DG. Intestine-specific gene transcription. Annu. Rev. Physiol. (1996) 58: 275-297
Traber PG. Differentiation of intestinal epithelial cells: Lessons from the study of intestine-specific gene expression. J Lab Clin Med. (1994)123: 467-477
Markowitz AJ, Wu GD, Birkenmeier EH, Traber PG. The human sucrase-isomaltase gene directs complex patterns of gene expression in transgenic mice. Am. J. Physiol. (1993) 265: G526-539
Rings EHHM, DeBoar PAJ, Moorman AFM, VanBeers EH, Deller J, et al. Lactase gene expression during early development of rat small intestine. Gastroenterology (1992) 103: 1154-1161
Hwang E-S, Hirayama BA, Wright EM. Distribution of the SGLT1 Na+/glucose cotransporter and mRNA along the crypt-villus axis of rabbit small intestine. Biochem. Biophys. Res. Commun. (1991) 181: 1208-1217
Noren O, Dabelsteen E, Hoyer PE, Olsen J, Sjostrom H, Hansen GH. Onset of transcription of the aminopeptidase N (leukemia antigen CD13) gene at the crypt/villus transition zone during rabbit enterocyte differentiation. FEBS Lett. (1989) 259: 107-112
Iseki S, Kondo H, Hitomi M, Ono T. Localization of liver fatty acid-binding protein and its mRNA in the liver and jejunum of rats: an immunohistochemical and in situ hybridization study. Mol. Cell Biochem. (1990) 98: 27-33
Rand EB, Depaoli AM, Davidson NO, bell GI, Burant CF. Sequence, tissue distribution, and functional characterization of the rat fructose transacterization of the rat fructose transporter GLUT5. Am. J. Physiol. (1993) 264: G1169-1176
Bookstein C, Depaoli AM, Xie Y, Niu P, Musch MW et al. Na+/H+ exchangers, NHE-1 and NHE-3, of rat intestine: expression and localization. J. Clin. Invest. (1994) 93: 106-113
Jin T, Drucker DJ. Activation of proglucagon gene transcription through a novel promoter element by the caudal-related homeodomain protein cdx-2/3. Mol. Cell Biol. (1996) 16(1): 19-28
Jin T, Trinh DK, Wang F, Drucker DJ. The caudal homeobox protein Cdx-2/3 activates endogenous proglucagon gene expression in InR1-G9 islet cells. Mol Endocrinol. (1997) 11(2): 203-209
Lambert M, Colnot S, Suh E, L'Horset F, Blin C, Calliot M E, Raymondjean M et al. Cis-Acting elements and transcription factors involved in the intestinal specific expression of the rat calbindin-D9K gene: binding of the intestine-specific transcription factor Cdx-2 to the TATA box. Eur J Biochem. (1996) 236(3): 778-788
Colnot S, Romagnolo B, Lambert M, Cluzeaud F, Porteu A, Vandewalle A, Thomasset M et al. Intestinal expression of the calbindin-D9K gene in transgenic mice. Requirement for a Cdx2-binding site in a distal activator region. J. Biol. Chem. (1998) 273(48): 31939-31946
Barley NF, Prathalingam SR, Zhi P, Legon S, Howard A, Walters JR. Factors involved in the duodenal expression of the human calbindin-D9k gene. Biochem. J. (1999) 341 ( Pt 3): 491-500
Drummond FJ, Sowden J, Morrison K, Edwards YH. The caudal-type homeobox protein Cdx-2 binds to the colon promoter of the carbonic anhydrase 1 gene. Eur. J. Biochem. (1996) 236(2): 670-681
Drummond FJ, Sowden J, Morrison K, Edwards YH. Colon carbonic anhydrase 1: transactivation of gene expression by the homeodomain protein Cdx2. FEBS Lett. (1998) 423(2): 218-222
Mitchelmore C, Troelsen JT, Spodsberg N, Sjostrom H, Noren O. Interaction between the homeodomain proteins Cdx-2 and HNF-1a mediates expression of the lactase-phlorizin hydrolase gene. Biochem. J. (2000) 346: 529-535
Sakaguchi T, Gu X, Golden HM, Suh E, Rhoads DB, Reinecker HC. Cloning of the human claudin-2 5’-flanking region revealed a TATA-less promoter with conserved binding sites in mouse and human for Caudal-Related Homeodomain Proteins and Hepatocyte Nuclear Factor-1a. J. Biol. Chem., epub. (2002)
Trinh KY, Jin T, and Drucker DJ. Identification of domains mediating transcriptional activation and cytoplasmic export in the caudal homeobox protein Cdx-3. J. Biol. Chem. (1999) 274: 6011-6019
Ho SN, Hunt HD, Horton RM, Pullen, JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene (1989) 77: 51-59
Boudreau F, Zhu Y, Traber PG. Sucrase-isomaltase gene transcription requires the hepatocyte nuclear factor-1 (HNF-1) regulatory element and is regulated by the ratio of HNF-1 alpha to HNF-1 beta. J. Biol. Chem. (2001) 276: 32122-32128
Silberg DG, Swain GP, Suh ER, Traber PG. Cdx1 and cdx2 expression during intestinal development. Gastroenterology (2000) 119: 961-971
Kuo CJ, Conley PB, Hsieh CL, Francke U, Crabtree GR. Molecular cloning, functional expression, and chromosomal localization of mouse hepatocyte nuclear factor 1. Proc. Natl. Acad. Sci. (1990) 87: 9838-9842
Serfas MS, Tyner AL. HNF-1 alpha and HNF-1 beta expression in mouse intestinal crypts. Am. J. Physiol. (1993) 265: 506-513
Boudreau F, Zhu Y, Traber PG. Sucrase-isomaltase gene transcription requires the hepatocyte nuclear factor-1 (HNF-1) regulatory element and is regulated by the ratio of HNF-1 alpha to HNF-1 beta. J. Biol. Chem. (2001) 276: 32122-32128
Levy E, Mehran M, Seidman E. Caco-2 cells as a model for intestinal lipoprotein synthesis and secretion. FASEB J. (1995) 9: 626-635
Suh E, Chen L, Taylor J, Traber PG. A homeodomain protein related to caudal regulates intestine-specific gene transcription. Mol Cell Biol. (1994) Nov;14(11): 7340-7351
Lambert M, Colnot S, Suh E, L'Horset F, Blin C, Calliot M E, Raymondjean M et al. Cis-Acting elements and transcription factors involved in the intestinal specific expression of the rat calbindin-D9K gene: binding of the intestine-specific transcription factor Cdx-2 to the TATA box. Eur J Biochem. (1996) 236(3): 778-788
Wu GD, Chen L, Forslund K, Traber PG. Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) and HNF-1 beta regulate transcription via two elements in an intestine-specific promoter. J Biol Chem. (1994) Jun 24;269(25): 17080-17085
Carey M. The enhanceosome and transcriptional synergy. Cell (1998) 92: 5-8
Darlington GJ. Molecular mechanisms of liver development and differentiation. Current Opinion in Cell Biology (1999)11: 678-682
Repa JJ, Mangelsdorf DJ. The role of orphane nuclear receptors in the regulation of cholesterol homeostasis. Annu. Rev. Cell Dev. (2000) 16: 459-481
Cordle SR, Whelan J, Henderson E, Masuoka H, Weil PA, Stein R. Insulin gene expression in nonexpressing cells appears to be regulated by multiple distinct negative-acting control elements. Mol. Cell Biol. (1991) 11: 2881-2886
Nir U, Walker MD, Rutter WJ. Regulation of rat insulin 1 gene expression: evidence for negative regulation in nonpancreatic cells. Proc. Natl Acad. Sci. USA (1986) 83: 3180-3184
Gerasimova TI, Corces VG. Chromatin insulators and boundaries: effects on transcription and nuclear organization. Annu Rev Genet. (2001) 35: 193-208
Mossuz P, Cousin F, Castinel A, Chauvet M, Sotto MF, Polack B, Sotto JJ, Kolodie L. Effects of two sex steroids (17beta estradiol and testosterone) on proliferation and clonal growth of the human monoblastic leukemia cell line, U937. Leuk Res. (1998) 22: 1063-1072
Tsuchiya S, Kobayashi Y, Goto Y, Okumura H, Nakae S, Konno T, Tada K. Induction of maturation in cultured human monocytic leukemia cells by a phorbol diester. Cancer Res. (1982) 42: 1530-1536
Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science (1980) 209: 497-499
Busch SJ, Barnhart RL, Martin GA, Flanagan MA, Jackson RL. Differential regulation of hepatic triglyceride lipase and 3-hydroxy-3- methylglutaryl-CoA reductase gene expression in a human hepatoma cell line, HepG2. J. Biol. Chem. (1990) 265: 22474-22479
Liao C, Zhao M, Song H, Uchida K, Yokoyama KK, Li T. Identification of the gene for a novel liver-related putative tumor suppressor at a high-frequency loss of heterozygosity region of chromosome 8p23 in human hepatocellular carcinoma. Hepatology (2000) 32: 721-727
Song BL, Qi W, Yang XY, Chang CC, Zhu JQ, Chang TY, Li BL. Organization of human ACAT-2 gene and its cell type-specific promoter activity. Biochem Biophys Res Commun. (2001) 282(2): 580-588
Yang JB, Duan ZJ, Yao W, Lee O, Yang L, Yang XY, Sun X, Chang CCY, Chang TY, Li BL. Synergistic Transcriptional Activation of Human ACAT-1 Gene by IFN- and ATRA in THP-1 cells. J. Biol. Chem. (2001) 276: 20989-20998
Liu J, Streiff R, Zhang YL, Vestal RE, Spence MJ, Briggs MR. Novel mechanism of transcriptional activation of hepatic LDL receptor by oncostatin M. J. Lipid Res. (1997) 38: 2035-2048
Powell JT, Klaasse JM Bos, Mourik JA. van. The uptake and expression of the factor VIII and reporter genes by vascular cells. FEBS Lett. (1992) 303: 173-177
Mack KD, Wei R, Elbagarri A, Abbey N, McGrath MS. A novel method for DEAE-dextran mediated transfection of adherent primary cultured human macrophages. J. Immunol. Methods (1998) 211: 79-86
Yeh HJ, Chu TH, Shen TW. Ultrastructure of Continuously Cultured Adult Human Liver Cell. Acta Biol. Exp. Sin. (1980) 13: 361-369
Lévy L, Neuveut C, Renard CA, Charneau P, Branchereau S, Gauthier F, Van Nhieu JT, Cherqui D, Petit-Bertron AF, Mathieu D, Buendia MA. Transcriptional Activation of Interleukin-8 by β-Catenin-Tcf4. J. Biol. Chem. (2002) 277: 42386-42393
Lee O, Chang CC, Lee W, Chang TY. Immunodepletion experiments suggest that acylcoenzyme A: cholesterol acyltransferase-1 (ACAT-1) protein plays a major catalytic role in adult human liver, adrenal gland, macrophages, and kidney, but not in intestines. J. Lipid Res. (1998) 39: 1722-1727
Sakashita N, Miyazaki A, Takeya M, Horiuchi S, Chang CCY, Chang TY, Takahashi K. Localization of Human Acyl-Coenzyme A:Cholesterol Acyltransferase-1(ACAT-1) in Macrophages and in Various Tissues. Am. J. Pathol. (2000) 156: 227-236
Sakashita N, Miyazaki A, Chang CC, Chang TY, Kiyota E, Satoh M, Komohara Y, Morganelli PM, Horiuchi S, Takeya M. Acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) is induced in monocyte-derived macrophages: in vivo and in vitro studies. Lab Invest (2003) 83: 1569-1581
Daniele B, Bencivenga A, Megna AS, Tinessa V. Alpha-fetoprotein and ultrasonography screening for hepatocellular carcinoma. Gastroenterology. (2004) 127(5 Suppl 1): S108-S112
Di Bisceglie AM. Issues in screening and surveillance for hepatocellular carcinoma. Gastroenterology. (2004) 127(5 Suppl 1): S104-S107
Marrero JA, Lok AS. Newer markers for hepatocellular carcinoma. Gastroenterology (2004) 127(5 Suppl 1): S113-S119
Ding SJ, Li Y, Tan YX, Jiang MR, Tian B, Liu YK, Shao XX, Ye SL, Wu JR, Zeng R, Wang HY, Tang ZY, Xia QC. From proteomic analysis to clinical significance: overexpression of cytokeratin 19 correlates with hepatocellular carcinoma metastasis. Mol. Cell Proteomics (2004) 3: 73-81
Merle P, de la Monte S, Kim M, Herrmann M, Tanaka S, Von Dem Bussche A, Kew MC, Trepo C, Wands JR. Functional consequences of frizzled-7 receptor overexpression in human hepatocellular carcinoma. Gastroenterology. (2004) 127: 1110-1122
Capurro M, Wanless IR, Sherman M, Deboer G, Shi W, Miyoshi E, Filmus J.Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology. (2003) 125: 89-97
Chang CCY, Sakashita N, Ornvold K, Lee O, Chang ET, Dong R, Lin S, Lee CY, Strom SC, Kashyap R, Fung JJ, Farese RV Jr, Patoiseau JF, Delhon A, Chang TY. Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine. J Biol Chem. (2000) 275(36): 28083-28092
Bennett CS, Tercyak AM. Reduced cholesterol transmucosal transport in rats inhibited mucosal acyl CoA: cholesterol acytransferase and normal pancreatic function. J. Lipid Res. (1984) 25: 148-159
Apergis GA, Crawford N, Ghosh D, Steppan CM, Vorachek MR, Wen P, Locker J. A novel nk-2-related transcription factor associated with human fetal liver and hepatocellular carcinoma. J. Biol. Chem. (1998) 273: 2917-2925
Li BL, Li XL, Duan ZJ, Lee O, Lin s, Ma ZM, Chang CC, Yang XY, Park JP, Mohandas TK, Noll W, Chan L, Chang TY. Human acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) gene organization and evidence that the 4.3-kilobase ACAT-1 mRNA is produced from two different chromosomes. J Biol Chem. (1999) 274(16):11060-11071
Chang CC, Huh HY, Cadigan KM, Chang TY et al. Molecular Cloning and Functional Expression of Human Acyl-Coenzyme A: Cholesterol Acyltransferase cDNA in Mutant Chinese Hamster Ovary Cells. J.Biol.Chem. (1993) 268: 20747-20755
Chang TY, Chang CCY, Chen D. Acyl-Coenzyme A: cholesterol acyltransferase. Ann. Rev. Biochem. (1996) 66: 613-638
Nilsen TW. Evolutionary origin of SL-addition trans-splicing: still an enigma. Trends Genet. (2001)17: 678-680
Denker JA, Zuckerman DM, Maroney PA, Nilsen TW. New components of the spliced leader RNP required for nematode trans-splicing. Nature (2002) 417: 667-670
Bruzik JP, Maniatis T. Spliced leader RNAs from lower eukaryotes are trans-spliced in mammalian cells. Nature (1992) 360: 692-695
Bruzik JP, Maniatis T. Enhancer-dependent interaction between 5' and 3' splice sites in trans. Proc. Natl. Acad. Sci. U S A. (1995) 92: 7056-7059
Chiara MD, Reed R. A two-step mechanism for 5' and 3' splice-site pairing. Nature (1995) 375: 510-513
Caudevilla C, Serra D, Miliar A, Codony C, Asins G, Bach M, Hegardt FG. Natural trans-splicing in carnitine octanoyltransferase pre-mRNAs in rat liver. Proc. Natl. Acad. Sci. U S A. (1998) 95: 12185-12190
Caudevilla C, Codony C, Serra D, Plasencia G, Roman R, Graessmann A, Asins G, Bach Elias M, Hegardt FG. Localization of an exonic splicing enhancer responsible for mammalian natural trans-splicing. Nucleic. Acids. Res. (2001) 29: 3108-3015
Troutt AB, McHeyzer-Williams MG, Pulendran B, Nossal GJV. Ligation-anchored PCR: A simple amplification technique with single-sided specificity. Proc. Natl. Acad. Sci. USA (1992) 89(20): 9823-9825
Troutt AB. Ligation-anchored PCR. In: Griffin AM, eds. Technology: current innovations. Boca Raton, Ann Arbor, London, Tokyo: CRC Press, (1994) 141-145
Anderson RA, Joyce C, Davis M, Reagan JW, Clark M, Shelness GS, Rudel LL. Identification of a form of acyl-CoA:cholesterol acyltransferase specific to liver and intestine in nonhuman primates. J. Biol. Chem. (1998) 273 (41): 26747-26754
Vellard M, et al. C-myb proto-oncogene: evidence for intermolecular recombination of coding sequences. Oncogene (1991) 6: 505-514
Sullivan PM, Petrusz P, Szpirer C, Joseph DR. Alternative processing of androgen-binding protein RNA transcripts in fetal rat liver. Identification of a transcript formed by trans splicing. J. Biol. Chem. (1991) 266: 143-154
Maniatis T, Tasic B. Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature (2002) 418: 236-243
Fairbrother WG, Yeh RF, Sharp PA, Burge CB. Predictive identification of exonic splicing enhancers in human genes. Science (2002) 297: 1007-1013
Cartegni L, Chew SL, Krainer AR. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat. Rev. Genet. (2002) 3: 285-298
Dorn R, Reuter G, Loewendorf A. Transgene analysis proves mRNA trans-spliced at the complex mod(mdg4) locus in Drosophila. Proc. Natl. Acad. Sci. USA (2001) 98: 9724-9729
