家族性急性髓系白血病cDNA消减文库的构建和差异表达基因的克隆、筛选及序列分析
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摘要
白血病是血液系统最常见的恶性肿瘤。白血病的发生是一个涉及多基因、多阶段的复杂过程,因此,全面分离与分析参与白血病发生的生物活性物质,揭示这些生物活性物质的功能与途径,对于系统研究与阐明白血病发生的分子机制尤为重要。遗传家系为我们提供了很好的样本来源,是获得疾病相关致病基因的一条重要途径。【目的】分离家族性急性髓系白血病患者骨髓中差异表达的基因,在基因水平上探讨急性白血病发生发展的分子机制。【方法】采用Super SMART和SSH 技术,以患者骨髓作为Tester,正常人骨髓作为Driver,分离患者骨髓细胞中差异表达的基因片段;连接T 载体,转化宿主菌,构建家族性急性髓系白血病cDNA 抑制性消减文库;采用DIG 标记探针的改良Differential Screening技术进行筛选和鉴定;阳性克隆送交测序,BLAST 同源性比对分析,新基因片段提交GenBank。【结果】成功构建了家族性急性髓系白血病cDNA 抑制性消减文库,巢式PCR 进一步使差异表达基因得到了指数扩增。产物连接载体并成功转化TOPO10F’大肠杆菌感受态细胞,使文库中的差异表达基因得到分离。经Differential Screening 技术筛选鉴定,确认28 个基因为阳性差异表达基因。测序结果提交GenBank 进行同源性比对分析,其中17 条与已知基因同源,11 条为未知基因片段。【结论】1. SSH 技术是筛选差异表达基因的有效方法。2. 采用DIG 标记探针替代同位素P~(32)-dCTP 标记探针,除继承了其高度灵敏度和良好的特异性之外,还避免了放射性污染,简化了实验操作。3. 获得多个与细胞增殖和分化相关的基因,说明急性白血病的发生是一个涉及多基因、多阶段发生的复杂过程。4. 获得11 条在文库中差异表达的新基因EST 片段,并被GenBank 收录,登录号分别为CX129926-7,CV884199,CV884202-3,CV973096-101。
Acute leukemia is a common malignant tumour of hematological system. It hasbeen proved by former researches that its pathogenesis relates to multi-genes andmulti-stages. However, it is still not enough evidence to explain it by current researchdata. So, isolating biological active substances in acute leukemia processes andrevealing their functional approaches are very important to understand molecularmechanisms of acute leukemia. The heredity families provide us the better samplesthat are helpful to understand the related virulence genes. 【Objective】To isolategenes expressed differentially from bone marrow cells of familial acute myelgenousleukemia and to explain the molecular mechanisms of the disease at the gene level.【Methods 】Super SMART cDNA synthesis and suppression subtractivehybridization (SSH) were performed to isolate differentially expressed cDNAfragments from patient’s bone marrow cells. cDNA from the patient were used as“tester”, the other from the normal human as “driver”. Subtractive products weredirectly inserted into T/A cloning vector, and then transformed into host bacteria, toset up a subtractive cDNA library of specially or highly expressed genes in familialacute myelogenous leukemia. Those clones were screened and identified by reformeddifferential screening technique which the probes were labeled with DIG. Positiveclones were sequenced and compared with known sequences in the public databasesof GenBank/EMBL/DDBJ using NCBI BLAST for homology analysis. The unknownfragments were then submitted to GenBank. 【Results】Total RNA were extractedand purified in good quality. Double strand cDNA were reverse transcriptedintegratedly, and then cut by RsaⅠinto even length short segments. Ligation wasidentified highly effective. After two hybridizations, a subtractive library of
differentially expressed genes in human familial acute myelgenous leukemia wasconstructed successfully by SSH. Nested-PCR was specificial, which amplified thedifferentially expressed genes exponentially. Subtractive products were inserted intothe T/A clone vectors and then transformed into TOPO10F’competent cellssuccessfully, which dispersed differentially expressed genes of the library into a singlecopy. After differential screening and RT-PCR, 28 clones were confirmed to bepositive differentially expressed genes fragments. Sequences were submitted toGenBank for homology analysis to determine what kind of protein they were encoding,and confirm which fragments were new ESTs. Then 17 sequences were a part ofknown genes, and the rests were verified unknown fragments.【Conclusion】(1) SSHis an effective approach to isolate differentially expressed genes. (2) Labeling probeswith DIG was applied in differential screening technology which substituted for theconventional P32-dCTP isotope probes. This technique was not only maintaining thehigh sensitivity and specificity but also averting isotope pollution and simplifyingexperimental procedures. (3) Some genes related to cell proliferation anddifferentiation were acquired by SSH, which proved sufficiently that the occurrence ofacute leukemia was involved in multi-genes and multi-stages. It is a complex process.(4) Eleven novel ESTs related to library were successfully isolated and accepted byGenBank, the access numbers are CX129926-7, CV884199, CV884202-3,CV973096-101, respectively.
Acute leukemia is a common malignant tumour of hematological system. It hasbeen proved by former researches that its pathogenesis relates to multi-genes andmulti-stages. However, it is still not enough evidence to explain it by current researchdata. So, isolating biological active substances in acute leukemia processes andrevealing their functional approaches are very important to understand molecularmechanisms of acute leukemia. The heredity families provide us the better samplesthat are helpful to understand the related virulence genes. 【Objective】To isolategenes expressed differentially from bone marrow cells of familial acute myelgenousleukemia and to explain the molecular mechanisms of the disease at the gene level.【Methods 】Super SMART cDNA synthesis and suppression subtractivehybridization (SSH) were performed to isolate differentially expressed cDNAfragments from patient’s bone marrow cells. cDNA from the patient were used as“tester”, the other from the normal human as “driver”. Subtractive products weredirectly inserted into T/A cloning vector, and then transformed into host bacteria, toset up a subtractive cDNA library of specially or highly expressed genes in familialacute myelogenous leukemia. Those clones were screened and identified by reformeddifferential screening technique which the probes were labeled with DIG. Positiveclones were sequenced and compared with known sequences in the public databasesof GenBank/EMBL/DDBJ using NCBI BLAST for homology analysis. The unknownfragments were then submitted to GenBank. 【Results】Total RNA were extractedand purified in good quality. Double strand cDNA were reverse transcriptedintegratedly, and then cut by RsaⅠinto even length short segments. Ligation wasidentified highly effective. After two hybridizations, a subtractive library of
differentially expressed genes in human familial acute myelgenous leukemia wasconstructed successfully by SSH. Nested-PCR was specificial, which amplified thedifferentially expressed genes exponentially. Subtractive products were inserted intothe T/A clone vectors and then transformed into TOPO10F’competent cellssuccessfully, which dispersed differentially expressed genes of the library into a singlecopy. After differential screening and RT-PCR, 28 clones were confirmed to bepositive differentially expressed genes fragments. Sequences were submitted toGenBank for homology analysis to determine what kind of protein they were encoding,and confirm which fragments were new ESTs. Then 17 sequences were a part ofknown genes, and the rests were verified unknown fragments.【Conclusion】(1) SSHis an effective approach to isolate differentially expressed genes. (2) Labeling probeswith DIG was applied in differential screening technology which substituted for theconventional P32-dCTP isotope probes. This technique was not only maintaining thehigh sensitivity and specificity but also averting isotope pollution and simplifyingexperimental procedures. (3) Some genes related to cell proliferation anddifferentiation were acquired by SSH, which proved sufficiently that the occurrence ofacute leukemia was involved in multi-genes and multi-stages. It is a complex process.(4) Eleven novel ESTs related to library were successfully isolated and accepted byGenBank, the access numbers are CX129926-7, CV884199, CV884202-3,CV973096-101, respectively.
引文
1 Gunz FW, Gunz JP, Veale AM, et al. Familial leukaemia: a study of 909 families. Scand J Haematol. 1975, 15:117-131.
2 Gunz FW, Gunz JP, Vincent PC, et al. Thirteen cases of leukemia in a family. J Natl Cancer Inst. 1978, 60:1243-1250.
3 梁玉英, 叶榆生, 卓光生, 等. 一个白血病高发家系的研究. 中华医学杂志, 1991, 71:428-430.
4 何立珍,唐南洪,吕联煌. 一个急性髓系白血病家系成员的染色体脆性部位观察. 中华医 学遗传学杂志. 1993, 10:359-360.
5 何立珍,张萍容,吕联煌. 一个白血病家系成员的SCE、Tc 及AgNOR 观察. 福建医学院 学报. 1993,27298-300.
6 He LZ, Lu LH, Chen ZZ. Genetic mechanism of leukemia predisposition in a family with 7 cases of acute myeloid leukemia. Cancer Genet Cytogenet. 1994, 76:65-69.
7 冯宝章, 雷健玲, 林泽嬉, 等. 一个急性髓系白血病家系的遗传学调查. 中华血液学杂志, 1991, 12:304-307.
8 Feng Baozhang, Lei Jianling, Lin Zexi, et al. Genetic Studies on a Famiky with Acute Myelogenous Leukemia. Cancer Genet Cytogent, 1999, 112:134-137.
9 Dyer MJ. The pathogenetic role of oncogenes deregulated by chromosomal translocation in B-cell malignancies. Int J Hematol. 2003, 77:315-320.
10 Alcalay M, Orleth A, Sebastiani C, et al. Common themes in the pathogenesis of acute myeloid leukemia. Oncogene. 2001, 20:5680-5694.
11 Rowley JD. The role of chromosome translocations in leukemogenesis. Semin Hematol. 1999, 36:59-72.
12 Golub TR. The genetics of AML: an update. In: Schechter GP, exec, ed. Hematology, 1999:American Society of Hematology Education program book:102-111.
13 贺林. 解码生命-人类基因组计划和后基因组计划. 北京: 科学出版社, 2004, 14-17.
14 Diatchenko L, Lau YC, Campbell AP, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93:6025-6030.
15 Diatchenko L, Lukyanow S, Lau YC, et al. Suppression subtractive hybridization: a versatile method for identifying differentially expressed genes. Methods Enzymol, 1999, 303:349-380.
16 Zhang J, Underwoods L E, D’Ercole A J. Formation of Chimeric cDNAs during suppression subtractive hybridization and subsequent polymerase chain reaction. Anal Biochem.
2000,282:259-262.
17 Akopyants NS, Fradkov A, Diatchenko L, et al. PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori. Proc Natl Acad Sci USA. 1998,95:13108-13113.
18 Endege WO, Steinmann KE, Boardman LA, et al. Representative cDNA libraries and their utility in gene expression profiling. Biotechniques.1999,26:542-548,550.
19 张成岗, 贺福出. 生物信息学在新基因全长cDNA 序列分析及功能预测中的应用. 生物化 学和生物物理进展. 2003,30:159-162.
20 吕军, 张颖, 冯立芹,等. 生物信息学工具BLAST 的使用. 内蒙古大学学报. 2003,34:181-187.
21 Negrutskii BS, El’skaya AV. Eukaryotic translation elongation factor 1 alpha: Structure, expression, functions, and possible role in aminoacyl-tRNA channeling. Prog Nucleic Acid Res. Mol. Biol. 1998, 60:47-78.
22 Morimoto H, Bonavida B. Diphtheria toxin and Pseudomonas A toxin-mediated apoptosis ADP ribosylation of elongation factor-2 is required for DNA fragmentation and cell lysis and synergy with tumor necrosis factor-alpha. J Immunol. 1992, 149:2089.
23 Shiina N, Gotoh Y, Kubomura N, et al. Microtubule severing by elongation factor 1 alpha. Science. 1994, 266:282.
24 Hitchens MR, Robbins PD. The role of the transcription factor DP in apoptosis. Apoptosis. 2003, 8:461-468.
25 Zhang Y, Venkatraj VS, Fischer SG, et al. Genomic cloning and chromosomal assignment of the E2F dimerization partner TFDP gene family. Genomics. 1997, 39:95-98.
26 Rogers KT, Higgins PD, Milla MM. DP-2, a heterodimeric partner of E2F: identification and characterization of DP-2 proteins expressed in vivo. Proc Natl Acad Sci USA. 1996, 93:7594-7599.
27 Wakasugi M, Kawashima A, Morioka H. DDB accumulates at DNA damage sites immediately after UV irradiation and directly stimulates nucleotide excision repair. J Biol Chem. 2002, 277:1637-1640.
28 Nichols AF, Itoh T, Zolezzi F. Basal transcriptional regulation of human damage-specific DNA-binding protein genes DDB1 and DDB2 by Sp1, E2F, N-myc and NF1 elements. Nucleic Acids Res. 2003, 31:562-569.
29 Nag A, Bondar T, Shiv S. The xeroderma pigmentosum group E gene product DDB2 is a specific target of cullin 4A in mammalian cells. Mol Cell Biol. 2001, 21:6738-6747.
30 Heung LJ, Del Poeta M. Unlocking the DEAD-box: a key to cryptococcal virulence? J Clin Invest. 2005, 115:593-595.
31 Kahlina K, Goren I, Pfeilschifter J. p68 DEAD box RNA helicase expression in keratinocytes. Regulation, nucleolar localization, and functional connection to proliferation and vascular endothelial growth factor gene expression. J Biol Chem. 2004, 279:44872-44882.
32 Coats S, Flanagan WM, Nourse J, et al. Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle. Science. 1996, 272:877-880.
33 Hunter T, Pines J. Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age. Cell. 1994, 79:573-582.
34 Toyoshima H, Hunter T. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell. 1994, 78:67-74.
35 Sherr CJ. Cancer cell cycles. Science. 1996, 274:1672-1677.
36 Kuerbitz SJ, Plunkett BS, Walsh WV, et al. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci USA. 1992, 89:7491-7495.
37 Kohn KW. Functional capabilities of molecular network components controlling the mammalian G1/S cell cycle phase transition. Oncogene. 1998, 16:1065-1075.
38 Poolman RA, Brooks G. Expressions and activities of cell cycle regulatory molecules during the transition from myocyte hyperplasia to hypertrophy. J Mol Cell Cardiol. 1998, 30:2121-2135.
39 Silverman GA, Bird PI, Carrell RW, et al. The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J Biol Chem. 2001, 276:33293-33296.
40 Sun J, Stephens R, Mirza G, et al. A serpin gene cluster on human chromosome 6p25 contains PI6, PI9 and ELANH2 which have a common structure almost identical to the 18q21 ovalbumin serpin genes. Cytogenet Cell Genet. 1998, 82:273-277.
41 Zeng W, Silverman GA, Remold-O'Donnell E. Structure and sequence of human M/NEI (monocyte/neutrophil elastase inhibitor), an Ov-serpin family gene. Gene. 1998, 213:179-187.
42 Cooley J, Takayama TK, Shapiro SD, et al. The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites. Biochemistry. 2001, 40:15762-15770.
43 Shur BD, Evans S, Lu Q. Cell surface galactosyltransferase: current issues. Glycoconj J. 1998, 15:537-548.
44 Wells L, Vosseller K, Hart GW. Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc. Science. 2001, 291:2376-2378.
45 Wu X, Rush JS, Karaoglu D, et al. Deficiency of UDP-GlcNAc:Dolichol Phosphate N-Acetylglucosamine-1 Phosphate Transferase (DPAGT1) causes a novel congenital disorder of Glycosylation Type Ij. Hum Mutat. 2003, 22:144-150.
46 Uechi T, Tanaka T, Kenmochi N. A complete map of the human ribosomal protein genes: assignment of 80 genes to the cytogenetic map and implications for human disorders. Genomics. 2001, 72:223-230.
47 Kim JH, You KR, Kim IH, et al. Over-expression of the ribosomal protein L36a gene is associated with cellular proliferation in hepatocellular carcinoma. Hepatology. 2004, 39:129-138.
48 Montano MM, Ekena K, Delage-Mourroux R, et al. An estrogen receptor-selective coregulator that potentiates the effectiveness of antiestrogens and represses the activity of estrogens. Proc Natl Acad Sci USA. 1999, 96:6947-6952.
49 Kurtev V, Margueron R, Kroboth K, et al. Transcriptional regulation by the repressor of estrogen receptor activity via recruitment of histone deacetylases. J Biol Chem. 2004, 279:24834-24843.
50 Wen YD, Perissi V, Staszewski LM, et al. The histone deaxetylase-3 complex contains nuclear receptor corepressors. Proc Natl Acad Sci USA. 2000, 97:7202-7207.
51 McDowell TL, Gibbons RJ, Sutherland H, et al. Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes. Proc Natl Acad Sci USA. 1999, 96:13983-13988.
52 Gibbons RJ, McDowell TL, Raman S, et al. Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation. Nat Genet. 2000, 24:368-371.
53 Tang J, Wu S, Liu H, Stratt R, et al. A novel transcription regulatory complex containing death domain-associated protein and the ATR-X syndrome protein. J Biol Chem. 2004, 279:20369-20377.
54 Tang P, Park DJ, Marshall Graves JA, et al. ATRX and sex differentiation. Trends Endocrinol Metab. 2004, 15:339-344.
2 Gunz FW, Gunz JP, Vincent PC, et al. Thirteen cases of leukemia in a family. J Natl Cancer Inst. 1978, 60:1243-1250.
3 梁玉英, 叶榆生, 卓光生, 等. 一个白血病高发家系的研究. 中华医学杂志, 1991, 71:428-430.
4 何立珍,唐南洪,吕联煌. 一个急性髓系白血病家系成员的染色体脆性部位观察. 中华医 学遗传学杂志. 1993, 10:359-360.
5 何立珍,张萍容,吕联煌. 一个白血病家系成员的SCE、Tc 及AgNOR 观察. 福建医学院 学报. 1993,27298-300.
6 He LZ, Lu LH, Chen ZZ. Genetic mechanism of leukemia predisposition in a family with 7 cases of acute myeloid leukemia. Cancer Genet Cytogenet. 1994, 76:65-69.
7 冯宝章, 雷健玲, 林泽嬉, 等. 一个急性髓系白血病家系的遗传学调查. 中华血液学杂志, 1991, 12:304-307.
8 Feng Baozhang, Lei Jianling, Lin Zexi, et al. Genetic Studies on a Famiky with Acute Myelogenous Leukemia. Cancer Genet Cytogent, 1999, 112:134-137.
9 Dyer MJ. The pathogenetic role of oncogenes deregulated by chromosomal translocation in B-cell malignancies. Int J Hematol. 2003, 77:315-320.
10 Alcalay M, Orleth A, Sebastiani C, et al. Common themes in the pathogenesis of acute myeloid leukemia. Oncogene. 2001, 20:5680-5694.
11 Rowley JD. The role of chromosome translocations in leukemogenesis. Semin Hematol. 1999, 36:59-72.
12 Golub TR. The genetics of AML: an update. In: Schechter GP, exec, ed. Hematology, 1999:American Society of Hematology Education program book:102-111.
13 贺林. 解码生命-人类基因组计划和后基因组计划. 北京: 科学出版社, 2004, 14-17.
14 Diatchenko L, Lau YC, Campbell AP, et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93:6025-6030.
15 Diatchenko L, Lukyanow S, Lau YC, et al. Suppression subtractive hybridization: a versatile method for identifying differentially expressed genes. Methods Enzymol, 1999, 303:349-380.
16 Zhang J, Underwoods L E, D’Ercole A J. Formation of Chimeric cDNAs during suppression subtractive hybridization and subsequent polymerase chain reaction. Anal Biochem.
2000,282:259-262.
17 Akopyants NS, Fradkov A, Diatchenko L, et al. PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori. Proc Natl Acad Sci USA. 1998,95:13108-13113.
18 Endege WO, Steinmann KE, Boardman LA, et al. Representative cDNA libraries and their utility in gene expression profiling. Biotechniques.1999,26:542-548,550.
19 张成岗, 贺福出. 生物信息学在新基因全长cDNA 序列分析及功能预测中的应用. 生物化 学和生物物理进展. 2003,30:159-162.
20 吕军, 张颖, 冯立芹,等. 生物信息学工具BLAST 的使用. 内蒙古大学学报. 2003,34:181-187.
21 Negrutskii BS, El’skaya AV. Eukaryotic translation elongation factor 1 alpha: Structure, expression, functions, and possible role in aminoacyl-tRNA channeling. Prog Nucleic Acid Res. Mol. Biol. 1998, 60:47-78.
22 Morimoto H, Bonavida B. Diphtheria toxin and Pseudomonas A toxin-mediated apoptosis ADP ribosylation of elongation factor-2 is required for DNA fragmentation and cell lysis and synergy with tumor necrosis factor-alpha. J Immunol. 1992, 149:2089.
23 Shiina N, Gotoh Y, Kubomura N, et al. Microtubule severing by elongation factor 1 alpha. Science. 1994, 266:282.
24 Hitchens MR, Robbins PD. The role of the transcription factor DP in apoptosis. Apoptosis. 2003, 8:461-468.
25 Zhang Y, Venkatraj VS, Fischer SG, et al. Genomic cloning and chromosomal assignment of the E2F dimerization partner TFDP gene family. Genomics. 1997, 39:95-98.
26 Rogers KT, Higgins PD, Milla MM. DP-2, a heterodimeric partner of E2F: identification and characterization of DP-2 proteins expressed in vivo. Proc Natl Acad Sci USA. 1996, 93:7594-7599.
27 Wakasugi M, Kawashima A, Morioka H. DDB accumulates at DNA damage sites immediately after UV irradiation and directly stimulates nucleotide excision repair. J Biol Chem. 2002, 277:1637-1640.
28 Nichols AF, Itoh T, Zolezzi F. Basal transcriptional regulation of human damage-specific DNA-binding protein genes DDB1 and DDB2 by Sp1, E2F, N-myc and NF1 elements. Nucleic Acids Res. 2003, 31:562-569.
29 Nag A, Bondar T, Shiv S. The xeroderma pigmentosum group E gene product DDB2 is a specific target of cullin 4A in mammalian cells. Mol Cell Biol. 2001, 21:6738-6747.
30 Heung LJ, Del Poeta M. Unlocking the DEAD-box: a key to cryptococcal virulence? J Clin Invest. 2005, 115:593-595.
31 Kahlina K, Goren I, Pfeilschifter J. p68 DEAD box RNA helicase expression in keratinocytes. Regulation, nucleolar localization, and functional connection to proliferation and vascular endothelial growth factor gene expression. J Biol Chem. 2004, 279:44872-44882.
32 Coats S, Flanagan WM, Nourse J, et al. Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle. Science. 1996, 272:877-880.
33 Hunter T, Pines J. Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age. Cell. 1994, 79:573-582.
34 Toyoshima H, Hunter T. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell. 1994, 78:67-74.
35 Sherr CJ. Cancer cell cycles. Science. 1996, 274:1672-1677.
36 Kuerbitz SJ, Plunkett BS, Walsh WV, et al. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci USA. 1992, 89:7491-7495.
37 Kohn KW. Functional capabilities of molecular network components controlling the mammalian G1/S cell cycle phase transition. Oncogene. 1998, 16:1065-1075.
38 Poolman RA, Brooks G. Expressions and activities of cell cycle regulatory molecules during the transition from myocyte hyperplasia to hypertrophy. J Mol Cell Cardiol. 1998, 30:2121-2135.
39 Silverman GA, Bird PI, Carrell RW, et al. The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J Biol Chem. 2001, 276:33293-33296.
40 Sun J, Stephens R, Mirza G, et al. A serpin gene cluster on human chromosome 6p25 contains PI6, PI9 and ELANH2 which have a common structure almost identical to the 18q21 ovalbumin serpin genes. Cytogenet Cell Genet. 1998, 82:273-277.
41 Zeng W, Silverman GA, Remold-O'Donnell E. Structure and sequence of human M/NEI (monocyte/neutrophil elastase inhibitor), an Ov-serpin family gene. Gene. 1998, 213:179-187.
42 Cooley J, Takayama TK, Shapiro SD, et al. The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites. Biochemistry. 2001, 40:15762-15770.
43 Shur BD, Evans S, Lu Q. Cell surface galactosyltransferase: current issues. Glycoconj J. 1998, 15:537-548.
44 Wells L, Vosseller K, Hart GW. Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc. Science. 2001, 291:2376-2378.
45 Wu X, Rush JS, Karaoglu D, et al. Deficiency of UDP-GlcNAc:Dolichol Phosphate N-Acetylglucosamine-1 Phosphate Transferase (DPAGT1) causes a novel congenital disorder of Glycosylation Type Ij. Hum Mutat. 2003, 22:144-150.
46 Uechi T, Tanaka T, Kenmochi N. A complete map of the human ribosomal protein genes: assignment of 80 genes to the cytogenetic map and implications for human disorders. Genomics. 2001, 72:223-230.
47 Kim JH, You KR, Kim IH, et al. Over-expression of the ribosomal protein L36a gene is associated with cellular proliferation in hepatocellular carcinoma. Hepatology. 2004, 39:129-138.
48 Montano MM, Ekena K, Delage-Mourroux R, et al. An estrogen receptor-selective coregulator that potentiates the effectiveness of antiestrogens and represses the activity of estrogens. Proc Natl Acad Sci USA. 1999, 96:6947-6952.
49 Kurtev V, Margueron R, Kroboth K, et al. Transcriptional regulation by the repressor of estrogen receptor activity via recruitment of histone deacetylases. J Biol Chem. 2004, 279:24834-24843.
50 Wen YD, Perissi V, Staszewski LM, et al. The histone deaxetylase-3 complex contains nuclear receptor corepressors. Proc Natl Acad Sci USA. 2000, 97:7202-7207.
51 McDowell TL, Gibbons RJ, Sutherland H, et al. Localization of a putative transcriptional regulator (ATRX) at pericentromeric heterochromatin and the short arms of acrocentric chromosomes. Proc Natl Acad Sci USA. 1999, 96:13983-13988.
52 Gibbons RJ, McDowell TL, Raman S, et al. Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation. Nat Genet. 2000, 24:368-371.
53 Tang J, Wu S, Liu H, Stratt R, et al. A novel transcription regulatory complex containing death domain-associated protein and the ATR-X syndrome protein. J Biol Chem. 2004, 279:20369-20377.
54 Tang P, Park DJ, Marshall Graves JA, et al. ATRX and sex differentiation. Trends Endocrinol Metab. 2004, 15:339-344.