1.Study on the genome structure of human telomeric repeat binding factor 1 and its pseudogenes.
Jie SUN ; He HUANG ; Huai-dong SONG ; Xin-yan WU
Journal of Zhejiang University. Medical sciences 2003;32(5):407-411
OBJECTIVETo determine the genome structure of human telomeric repeat binding factor 1 (TERF1) and its pseudogenes.
METHODSSequences were obtained from GenBank and analyzed using the BLAST program and other relevant biology program (Sequencher, DNA Strider and Autoassembler, etc) to determine the genome and pseudogenome structure of TERF1. PCR and sequencing were performed to verify the results.
RESULTTERF1 gene which mapped to 8q13 was divided into 10 exons. It had four processed pseudogenes located on chromosome 13, 18, 21 and X respectively (Psi TERF1-13 Psi TERF1-18 Psi TERF1-21 and Psi TERF1-X ). They were entire intronless TERF1 genes which lacked some exons. Three homologous fragments of at least 60 kb on the flanking region of Psi TERF1-13, Psi TERF1-18 and Psi TERF1-21, respectively were noted.
CONCLUSIONTERF1 gene has 10 exons. It has four processed pseudogenes which are located on chromosome 13, 18, 21, and X, respectively. Large homologous fragments that belong to the recently duplicated segments are transchromosomal duplications.
Chromosome Mapping ; Genetic Structures ; Humans ; Pseudogenes ; Telomeric Repeat Binding Protein 1 ; genetics
2.Research progress on telomere binding proteins.
Journal of Zhejiang University. Medical sciences 2004;33(6):469-473
Apoptosis
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physiology
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Cell Cycle
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physiology
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Humans
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Neoplasms
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etiology
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metabolism
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Tankyrases
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genetics
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physiology
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Telomerase
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metabolism
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physiology
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Telomere
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genetics
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metabolism
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Telomere-Binding Proteins
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genetics
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physiology
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Telomeric Repeat Binding Protein 1
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genetics
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physiology
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Telomeric Repeat Binding Protein 2
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genetics
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physiology
3.Isolation of Tara protein and its gene cloning.
Jian-ping LAN ; Yi LUO ; Yuan-yuan ZHU ; Jie SUN ; Xiao-yu LAI ; Jing-yuan LI ; Jian YU ; Ji-min SHI ; Mao-fang LIN ; He HUANG
Journal of Zhejiang University. Medical sciences 2004;33(6):486-490
OBJECTIVETo isolate and identify TRF1 immunoprecipitating protein complex and to clone the candidate gene.
METHODSThe co-immunoprecipitation assay was employed to isolate TRF1 protein complex and the immunoprecipitate was subjected to MALDI-TOF mass spectrometry for protein identification. The candidate gene was amplified by temperature-gradient PCR from human testis cDNA library and then cloned into pEGFP-C2 vector for eukaryotic expression. The amplified gene was verified by direct sequencing and GFP-tagged protein was confirmed by immunoblotting.
RESULTSTara protein with the size of 68 kD was identified from the TRF1 precipitate. The candidate gene amplified from cDNA library was about 1.7 kb as expected. Sequencing demonstrated the amplified fragment had 99.9% of homogenesis with Tara CDS sequence (gi:30474869). GFP-tagged fusion protein was about 100 kD. Tara was diffusely distributed in cytoplasm at interphase and in whole cells at mitotic phase.
CONCLUSIONTara might be an interacting protein with TRF1. However, further investigation would be required to confirm if they were bona fide partners.
Cloning, Molecular ; HeLa Cells ; Humans ; Microfilament Proteins ; genetics ; isolation & purification ; metabolism ; Protein Binding ; Telomeric Repeat Binding Protein 1 ; chemistry ; genetics ; metabolism
4.Preparation and characterization of monoclonal antibody against human telomeric repeat binding factor 1.
He HUANG ; Jimin SHI ; Qiaofang CHEN ; Yi LUO ; Wei DING ; Jiyu LOU
Chinese Journal of Hematology 2002;23(12):631-633
OBJECTIVETo prepare a monoclonal antibody against human telomeric repeat binding factor 1 (TRF1) protein and explore its biological characteristics.
METHODSBALB/c mice were immunized with GST-TRF1(33-277) fusion protein for the preparation of monoclonal antibody by hybridoma technique. The obtained antibody was used for clinical assay by Western-blot and immunohistochemical staining.
RESULTSOne strain of hybridoma was obtained. It was confirmed by Western-blot that the antibody specifically recognized the 60 kD TRF1 protein. Immunohistochemical staining of the antibody showed that TRF1 protein located in the cytoplasm of epithelial cells and bone marrow cells.
CONCLUSIONA TRF1 monoclonal antibody, with high specificity was developed. It is useful for detection of TRF1 protein in tissue specimens.
Animals ; Antibodies, Monoclonal ; immunology ; Blotting, Western ; Female ; Humans ; Hybridomas ; immunology ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; immunology ; Telomeric Repeat Binding Protein 1 ; analysis ; genetics ; immunology
5.Expression of TRF1, TRF2 and RAP1 mRNA in peripheral blood mononuclear cells of patients with acquired aplastic anemia.
Yan WANG ; Rui-Rong XU ; Yan-Feng ZHOU ; Zhao-Xia LIU
Journal of Experimental Hematology 2012;20(5):1158-1161
This study was aimed to investigate the expression levels of TRF1, TRF2 and RAP1 mRNA in peripheral blood mononuclear cells of patients with acquired aplastic anemia, and to explore their relation with onset of acquired aplastic anemia. Peripheral blood mononuclear cells of 40 patients with acquired aplastic anemia and 20 normal subjects as control were collected to detect mRNA expression of TRF1, TRF2 and RAP1 by using real-time quantitative polymerase chain reaction. The results showed that the expression levels of TRF1 and RAP1 in peripheral blood mononuclear cells of patients with acquired aplastic anemia were significantly higher than that in normal controls (P < 0.05), while the expression level of TRF2 was lower than that in normal controls (P < 0.01). There was significant correlation between TRF2 and RAP1 expressions level (r = 0.522, P = 0.001). It is concluded that the changes in expression levels of TRF1, TRF2 and RAP1 may play a role in the pathogenesis of acquired aplastic anemia.
Adolescent
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Adult
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Aged
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Aged, 80 and over
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Anemia, Aplastic
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blood
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Case-Control Studies
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Child
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Female
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Humans
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Leukocytes, Mononuclear
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metabolism
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Male
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Middle Aged
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RNA, Messenger
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genetics
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Telomeric Repeat Binding Protein 1
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metabolism
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Telomeric Repeat Binding Protein 2
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metabolism
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Young Adult
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rap1 GTP-Binding Proteins
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metabolism
6.Localization of human telomere repeat binding factor 1 in telomerase-positive and-negative cells and its expression during cell cycle.
Jian-ping LAN ; Xiao-yu LA ; Yuan-yuan ZHU ; Jie SUN ; Jing-yuan LI ; Jian YU ; Ya-min TAN ; Ji-min SHI ; Mao-fang LIN ; He HUANG
Journal of Zhejiang University. Medical sciences 2004;33(6):475-495
OBJECTIVETo observe the distribution pattern of human telomere repeat binding factor 1(TRF1) in the telomerase-positive (HeLa) and telomerase-negative cells (WI38-2RA) and to investigate its expression level during the cell cycle.
METHODSThe full-length sequences of TRF1(TRF1FL) and its mutant with N and C terminus deletion (TRF1DeltaNC) were generated by PCR amplification, the resulting fragments were cloned into pEGFP-C2 mammalian expression vector. GFP-tagged proteins were verified by Western blotting with rabbit anti-TRF1 and mouse anti-GFP antibodies after cell transfection. Immunofluorescence staining were performed to detect the TRF1 localization in HeLa and WI38-2RA cells. Metaphase spreads from HeLa cells were also prepared to observe TRF1 localization in chromosomes. HeLa cells were arrested by thymidine and nocodazole at different cell stages. Cell cycles were analyzed by flow cytometry and TRF1 levels were evaluated by semi-quantitative Western blotting.
RESULTSTRF1FL and TRF1PNC fragments were sized about 1.3 kb and 0.95 kb. GFP-tagged TRF1FL and TRF1DeltaNC proteins were 80 kD and 60 kD, respectively. In both HeLa and WI38-2RA cells, TRF1FL had a speckled distribution in the nuclei,however, TRF1FL did not coincide with promyelocytic leukemia (PML) nuclear body in HeLa cells while it exclusively did in WI38-2RA cells. Moreover, TRF1FL was exactly localized at the termini of metaphase spreads in HeLa cells. In contrast, TRF1PNC was diffusely distributed throughout the nuclei. Analysis by semi-quantitative Western blotting indicated that TRF1 levels increased with cell cycle progression, which reached the zenith at the M phase and went down to the nadir at G1/S point. The TRF1 level at M phase was about 3.9 times than that at G1/S point(t=12.92iP<0.01).
CONCLUSIONTRF1 has a different localization in telomerase-positive and telomerase-negative cells, which suggests TRF1 might exert different functions in these cells. TRF1 level is regulated with cell cycle.
Cell Cycle ; HeLa Cells ; Humans ; Leukemia, Promyelocytic, Acute ; pathology ; Mutation ; Telomerase ; metabolism ; Telomere-Binding Proteins ; biosynthesis ; genetics ; metabolism ; Telomeric Repeat Binding Protein 1 ; biosynthesis ; genetics ; metabolism ; Tumor Cells, Cultured
7.Expression of telomere repeat binding factor 1 (TRF1) protein in kidney cancer.
Ji-min SHI ; Wei DING ; He HUANG ; Zhi-gen ZHANG ; Li-dan TANG ; Mao-fang LIN
Journal of Zhejiang University. Medical sciences 2004;33(6):496-508
OBJECTIVETo investigate the expression levels of telomere repeat binding factor 1(TRF1) protein in normal kidney tissue and kidney cancer.
METHODSSpecimens of kidney cancer and pericancerous tissues were collected from 32 cases of renal carcinoma. A quantitative Western blotting technique was developed using TRF1 monoclonal antibody to determine the expression level of TRF1 protein in total protein extracts from tissue specimens.
RESULTSThe expression level of TRF1 protein was higher in normal kidney tissues (3.611 +/-1.922 microg/microl) than that of cancer tissues (2.428 +/-1.352 microg/microl) (t=5.776, P<0.01).
CONCLUSIONThe expression level of TRF1 protein is significantly reduced in kidney cancer and the level is negatively correlated with malignant degree of the cancer.
Adenocarcinoma, Clear Cell ; metabolism ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Kidney ; metabolism ; Kidney Neoplasms ; metabolism ; Male ; Middle Aged ; RNA, Messenger ; biosynthesis ; genetics ; Telomeric Repeat Binding Protein 1 ; biosynthesis ; genetics
8.Expression of human telomere repeat binding factor 1 (TRF1) in acute leukemia cells and its correlation with telomerase activities.
Jie SUN ; Xiao-yu LAI ; Yuan-yuan ZHU ; Jian-ping LAN ; Li-dan TANG ; Jing-yuan LI ; Jian YU ; Ya-min TAN ; Mao-fang LIN ; He HUANG
Journal of Zhejiang University. Medical sciences 2004;33(6):491-495
OBJECTIVETo study the expression of human telomere repeat binding factor 1 (TRF1) to investigate the correlation of telomerase activity with acute leukemia.
METHODSLeukemic cells were collected from 30 cases of acute leukemia. Realtime quantitative PCR with fluorescence probe hybridization was used to measure expression of TRF1 and hTERT mRNA in leukemic cells.
RESULTSTRF1 mRNA expression was 0.0126 (0.0127-0.0546) in acute non-lymphocytic leukemia (ANLL), which was lower than that in normal mononuclear cells [0.0457 (0.00839-0.262), P<0.001], but its expression in acute lymphoblastic leukemia (ALL) cells [0.0745 (1.92 x 10(-6)-0.193)] had no significant difference compared with that in normal mononuclear cells. TRF1 expression in ANLL cells was significantly lower than that in ALL cells (P=0.001). The expressions of TRF1 mRNA in AL cells and normal mononuclear cells had no significant correlation with expression of hTERT mRNA (r=-0.173, P=0.207).
CONCLUSIONThe expression of TRF1 is lower in ANLL cells, which indicates TRF1 may have some effect on telomerase activity by regulating telomere length in ANLL cells.
Adolescent ; Adult ; Aged ; Female ; Humans ; Leukemia, Myeloid, Acute ; enzymology ; metabolism ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; enzymology ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Telomerase ; metabolism ; Telomeric Repeat Binding Protein 1 ; biosynthesis ; genetics
9.Cloning and expression of hTRF1 in Escherichia coli and preparation of polyclonal antibody.
Hong JIANG ; Xiao-Fei ZHENG ; Ying LUO ; Jie ZHU ; Han-Jiang FU ; Qiang-Ling SUN ; Chang-Hao CHEN ; Zhi-Xian SUN
Chinese Journal of Biotechnology 2004;20(1):30-33
Human telomeric repeat binding factor 1(TRF1) contains one Myb-type DNA-binding repeat and an amino-terminal acidic domain. It can bind to the duplex array of TTAGGG repeats at chromosome ends and is shown to be important in preserving genomic stability, maintaining cell proliferative capacity, and blocking the activation of DNA-damage cell cycle checkpoints. Interestingly, the double strand DNA breaks sensor ATM interacts with and phosphorylates Pin2/TRF1 and inhibits its function after DNA damage. Are there some proteins else that can interact with TRF1 and influence its function? In order to analysis the interaction between TRF1 and other proteins, we must prepare the antiserum that can recognize the endogenous TRF1 of cell lysates. TRF1 cDNA was amplified using cDNA Library of HeLa cell by PCR and cloned into pUCm-T vector. Sequence analysis reveals identity to the GenBank report. The TRF1 cDNA was subcloned into expression vector pET-28c(+) and expressed in E. coli as a fusion protein of 65 kD. The recombinant TRF1 can express in the supernatant (about 12.3% in total protein) on the induction of 0.5 mmol/L IPTG at 37 degrees C for 3 hours. Western-blot analysis showed the recombinant protein can react with TRF1 polyclonal antibody sc-6165 (from Santa Cruz Company). His6-TRF1 was purified by Ni(2+) -NTA resin affinity chromatography made by ourselves and showed to be homogeneity in SDS-PAGE. Rabbits were immunized for four times to prepare polyclonal antibody. The unpurified antiserum can recognize the overexpressed TRF1 with myc-tag and the endogenous Pin2/TRF1 of cell lysate by Western-blot at 1:1000 dilution. At 1:400 dilution, the antiserum can interact with endogenous TRF1 by Immunofluorescence cell staining analysis. The endogenous TRF1 in different cell lines, such as HepG2, 803, MCF7 and HeLa, locates in the nucleus. The soluble expression TRF1 and preparation of its antibody lay the foundation to study it further.
Animals
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Antibodies
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immunology
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Cloning, Molecular
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Escherichia coli
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genetics
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HeLa Cells
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Humans
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Immune Sera
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immunology
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Rabbits
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Recombinant Fusion Proteins
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biosynthesis
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immunology
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isolation & purification
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Telomeric Repeat Binding Protein 1
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genetics
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immunology
10.Expression level of TRF1 protein in human acute leukemia and its relationship with activity of telomerase.
Ji-Min SHI ; He HUANG ; Qiao-Fang CHEN ; Mao-Fang LIN
Journal of Experimental Hematology 2006;14(5):858-861
The study was aimed to investigate the expression level of TRF1 protein in human acute leukemia and relationship between expression level of TRF1 protein and activity of telomerase. A quantitative Western blot technique was developed using anti-TRF1(33 - 277) monoclonal antibody and GST-TRF1 fusion protein as a standard to further determine the expression level of TRF1 protein in total proteins extracted from clinical specimens. 20 cases of acute leukemias were studied when 11 normal volunteer's bone marrow was used as control. The results showed that the expression level of TRF1 protein in normal bone marrow (2.217 +/- 0.461 microg/microl) was significantly higher than that in bone marrow of acute leukemia patients (0.754 +/- 0.343 microg/microl) (P < 0.01). There was no remarkable difference of expression level of TRF1 protein between ALL and ANLL (0.628 +/- 0.281 microg/microl vs 0.844 +/- 0.360 microg/microl, P > 0.05). After chemotherapy, TRF1 expression level of patients with complete remission raised (0.772 +/- 0.307 microg/microl vs 1.683 +/- 0.344 microg/microl, P < 0.01), but lower than that of normal (2.217 +/- 0.461 microg/microl, P < 0.01). TRF1 expression level of patients without complete remission was not remarkable different after chemotherapy (0.726 +/- 0.443 microg/microl vs 0.894 +/- 0.338 microg/microl, P > 0.05). TRF1 expression level of patients with complete remission was higher than that in patients without complete remession (1.683 +/- 0.344 microg/microl vs 0.894 +/- 0.338 microg/microl, P < 0.01). For all sample the telomerase activity was determined. It was confirmed that the activity of telomerase in normal bone marrow was lower than that in bone marrow of acute leukemia patients (0.125 +/- 0.078 microg/microl vs 0.765 +/- 0.284 microg/microl, P < 0.01). There was no significantly difference of expression level of TRF1 protein between ALL and ANLL (0.897 +/- 0.290 microg/microl vs 0.677 +/- 0.268 microg/microl, P > 0.05). After chemotherapy, telomerase activity of patients with complete remission reduced (0.393 +/- 0.125 microg/microl), but higher than that of normal (0.125 +/- 0.078 microg/microl, P < 0.01). It is concluded that expression level of TRF1 protein in AL patients is significantly decrese and associated with therapeutic efficaciousness and the activity of telomerase (P < 0.001).
Adolescent
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Adult
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Aged
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Bone Marrow Cells
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enzymology
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metabolism
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Child
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Female
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Humans
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Leukemia, Myeloid, Acute
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enzymology
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metabolism
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Male
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Middle Aged
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Precursor Cell Lymphoblastic Leukemia-Lymphoma
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enzymology
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metabolism
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Telomerase
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metabolism
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Telomeric Repeat Binding Protein 1
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biosynthesis
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genetics