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1.
Leukemia ; 37(5): 988-1005, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37019990

RESUMO

Chromosomal rearrangements of the human KMT2A/MLL gene are associated with de novo as well as therapy-induced infant, pediatric, and adult acute leukemias. Here, we present the data obtained from 3401 acute leukemia patients that have been analyzed between 2003 and 2022. Genomic breakpoints within the KMT2A gene and the involved translocation partner genes (TPGs) and KMT2A-partial tandem duplications (PTDs) were determined. Including the published data from the literature, a total of 107 in-frame KMT2A gene fusions have been identified so far. Further 16 rearrangements were out-of-frame fusions, 18 patients had no partner gene fused to 5'-KMT2A, two patients had a 5'-KMT2A deletion, and one ETV6::RUNX1 patient had an KMT2A insertion at the breakpoint. The seven most frequent TPGs and PTDs account for more than 90% of all recombinations of the KMT2A, 37 occur recurrently and 63 were identified so far only once. This study provides a comprehensive analysis of the KMT2A recombinome in acute leukemia patients. Besides the scientific gain of information, genomic breakpoint sequences of these patients were used to monitor minimal residual disease (MRD). Thus, this work may be directly translated from the bench to the bedside of patients and meet the clinical needs to improve patient survival.


Assuntos
Histona-Lisina N-Metiltransferase , Leucemia Mieloide Aguda , Proteína de Leucina Linfoide-Mieloide , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Lactente , Pré-Escolar , Criança , Adolescente , Adulto , Pessoa de Meia-Idade , Idoso , Leucemia Mieloide Aguda/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Fusão Gênica
2.
Leukemia ; 32(2): 273-284, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28701730

RESUMO

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.


Assuntos
Histona-Lisina N-Metiltransferase/genética , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Adulto , Criança , Aberrações Cromossômicas , Quebra Cromossômica , Feminino , Rearranjo Gênico/genética , Humanos , Lactente , Masculino , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética/genética
3.
Leukemia ; 27(11): 2165-76, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23628958

RESUMO

Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79 TPGs are now characterized at the molecular level. However, only seven rearrangements seem to be predominantly associated with illegitimate recombinations of the MLL gene (≈ 90%): AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, ELL, partial tandem duplications (MLL PTDs) and MLLT4/AF6, respectively. The MLL breakpoint distributions for all clinical relevant subtypes (gender, disease type, age at diagnosis, reciprocal, complex and therapy-induced translocations) are presented. Finally, we present the extending network of reciprocal MLL fusions deriving from complex rearrangements.


Assuntos
Quebra Cromossômica , Rearranjo Gênico , Leucemia/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Translocação Genética/genética , Doença Aguda , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Animais , Criança , Pré-Escolar , Feminino , Histona-Lisina N-Metiltransferase , Humanos , Lactente , Recém-Nascido , Leucemia/classificação , Masculino , Camundongos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Adulto Jovem
4.
Phytochemistry ; 49(2): 403-11, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9747538

RESUMO

Cell suspension cultures of Ruta graveolens L. accumulate polyketide metabolites such as acridone alkaloids and flavonoid pigments. Whereas flavonoid synthesis is induced by light, the production of alkaloids can be enhanced in dark-cultured cells by treatment with fungal elicitors. Acridone synthase (ACS) catalyzes the committed condensing reaction of acridone biosynthesis yielding 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl- and malonyl-CoAs. The reaction proceeds in a manner analogous to that of chalcone synthase (CHS) which catalyzes the first committed step in flavonoid biosynthesis and cDNA and protein sequences of Ruta ACS possess a high degree of sequence homology to heterologous CHSs. ACS transcript abundance and specific activity were monitored in cultured R. graveolens cells irradiated either continuously with white light or treated with fungal elicitor over a period of 24 h and found to increase transiently upon elicitor treatment and to decrease upon light irradiation. Immunodetection with a rabbit polyclonal ACS antiserum revealed that the amounts of ACS polypeptide decreased slightly in light-irradiated cells but increased in elicitor-treated Ruta cells. Fluorescence microscopy and tissue print hybridizations were employed to aid in localizing the sites of storage and biosynthesis of acridone alkaloids in Ruta plants. Yellow fluorescing alkaloids were detected particularly in root tissue adjacent to the rhizodermis, but also in the endodermis and vascular tissue of the hypocotyl. ACS transcript abundance in situ followed the same spatial pattern, indicating that the synthesis of acridones likely proceeds at all sites of deposition rather than exclusively in the root. Expression in planta and the induction response of ACS suggest that the alkaloids serve as phytoanticipins or phytoalexins in the defense of Ruta particularly to soil-borne pathogens or as feeding deterrents.


Assuntos
Aciltransferases/metabolismo , Proteínas de Plantas/metabolismo , Plantas/enzimologia , Acridinas/metabolismo , Acridonas , Aciltransferases/biossíntese , Aciltransferases/genética , Animais , Células Cultivadas , DNA de Plantas/metabolismo , Plantas/efeitos da radiação , RNA Mensageiro/metabolismo , Coelhos , Distribuição Tecidual , Transcrição Gênica
5.
Plant Mol Biol ; 27(4): 681-92, 1995 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7727746

RESUMO

Cell suspension cultures of Ruta graveolens L. produce a variety of acridone alkaloids, and the accumulation can be stimulated by the addition of fungal elicitors. Acridone synthase, the enzyme catalyzing the synthesis of 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl-CoA and malonyl-CoA, had been isolated from these cells, and the partial enzyme polypeptide sequence, elucidated from six tryptic fragments, revealed homology to heterologous chalcone synthases. Poly(A)+ RNA was isolated from Ruta cells that had been treated for 6 h with a crude cell wall elicitor from Phytophthora megasperma f. sp. glycinea, and a cDNA library was constructed in lambda 2AP. Clones harboring acridone synthase cDNA were isolated from the library by screening with a synthetic oligonucleotide probe complementary to a short stretch of sequence of the enzyme peptide with negligible homology to chalcone synthases. The identity of the clones was substantiated by DNA sequencing and by recognition of five additional peptides, determined previously from tryptic acridone synthase digests, in the translated sequence. An insert of roughly 1.4 kb encoded the complete acridone synthase, and alignments at both DNA and protein levels corroborated the high degree of homology to chalcone synthases. Expression of the enzyme in vector pET-11c in the Escherichia coli pLysS host strain proved the identity of the cloned cDNA. The heterologous enzyme in the crude E. coli extract exhibit high acridone but no chalcone synthase activity. The results were fully supported by northern blot hybridizations which revealed that the specific transcript abundance did not increase but rather decreased upon white light irradiation of cultured Ruta graveolens L. cells, a condition that commonly induces the abundance of chalcone synthase transcripts.


Assuntos
Aciltransferases/genética , Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Clonagem Molecular , DNA Complementar , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Células Vegetais , Plantas/enzimologia , Alinhamento de Sequência
6.
Z Naturforsch C J Biosci ; 49(1-2): 26-32, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-8148006

RESUMO

Acridone synthase has been purified from cell suspension cultures of Ruta graveolens using a combination of gel filtration and ion exchange chromatography. The purified enzyme has an apparent molecular weight of 69 kDa on gel filtration and a subunit structure on SDS-PAGE of 40 kDa. The apparent Km-values are 10.64 microM and 32.8 microM for N-methylanthraniloyl-CoA and malonyl-CoA, respectively. Tryptic digestion of the homogeneous acridone synthase was performed. Seven of the peptides were chosen for microsequencing. The homology of the amino acid sequences from this particular polypeptide and corresponding peptides from chalcone synthase 3 from garden pea amounted to 76%.


Assuntos
Aciltransferases/química , Aciltransferases/isolamento & purificação , Plantas/enzimologia , Aciltransferases/metabolismo , Sequência de Aminoácidos , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Homologia de Sequência de Aminoácidos , Tripsina
7.
Planta Med ; 48(8): 258-62, 1983 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17404992

RESUMO

Cell-free extracts from acridone synthesizing cell suspension cultures of RUTA GRAVEOLENS L. catalyze the N-methylation of anthranilic acid using S-adenosyl-L-methionine as methyl donor. The stability of enzyme preparations was remarkably high during storage at -20 degrees C. Optimum activity was exhibited at pH 8.2, Mg (2+) was not required for maximum activity and EDTA did not affect the reaction rate. The rate of N-methylanthranilic acid formation was shown to be linear for about 45 min and was proportional to the protein concentration up to at least 0.350 mg of the enzyme preparation. In a number of suspension cultures of plant species not belonging to the Rutaceae this particular N-methyltransferase was not found. Apparantly N-methylation of anthranilic acid is the first pathway-specific reaction in acridone alkaloid biosynthesis.

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