RESUMO
A PCR-based strategy was used to isolate a 2653 bp cDNA encoding the mouse sodium-dependent, purine nucleoside selective, concentrative nucleoside transporter (designated mCNT2). The deduced protein sequence exhibits 93 and 80% identity to the previously cloned rat and human sodium-dependent, purine nucleoside selective, nucleoside transporters, respectively. Characterization of 3H-nucleoside uptake by COS-1 cells transiently transfected with the cDNA demonstrated that it encoded a functional nucleoside transport activity with selectivity for purine nucleosides. The cDNA was used to screen a murine (strain 129SvJ/6) genomic library in pBeloBAC11 to identify a clone containing the mCNT2 gene. A PCR strategy was used to identify and sequence the intron-exon boundaries and to determine the approximate sizes of the introns. The mCNT2 gene spans approximately 13.7 kb and is encoded by 15 exons. The gene was mapped to mouse chromosome 2e3 by fluorescence in situ hybridization.
Assuntos
Proteínas de Transporte/genética , Genes/genética , Proteínas de Membrana Transportadoras , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS/metabolismo , Linhagem Celular , Mapeamento Cromossômico , Cromossomos/genética , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Éxons , Formicinas/farmacocinética , Expressão Gênica , Hibridização in Situ Fluorescente , Íntrons , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Nucleosídeos/farmacocinética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , TransfecçãoRESUMO
This study surveyed strategies of sequencing primer selection and evaluated primer performance in automated DNA sequencing. We asked participants to relate their preferred primer design strategies to identify primer characteristics that are considered most important in sequencing primer design. The participants preferred primers of 18-24 nucleotides (nt), 39%-58% G + C, a melting temperature (Tm) of 53 degrees-65 degrees C with a 1-2 nt 3' GC clamp, hairpin stems of less than 2-3 bp, homopolymeric runs of less than 4-5 nt, primer dimers of less than 3-4 bp and secondary priming sites of less than 3-4 bp. We provided a 300-bp test sequence and asked participants to submit sequences of 1-3 optimal sequencing primers. Submitted primers ranged from 17-24 nt and largely conformed to the preferred parameters. Submitted primers were distributed across the test sequence, although some sites were disfavored. Surprisingly, approximately 45% of the primers were selected "manually", more than by any software package. Each of 69 submitted and 95 control primers, distributed at 3-bp intervals across the test sequence, were synthesized, purified and tested using a Model 377 PRISM DNA Sequencer with dichlororhodamine dye terminator reagents (dRhodamine dye terminators). Approximately half of the control primers were also tested using rhodamine dye terminator reagents ("old" rhodamine dye terminators). The results indicated that primer physico-chemical characteristics thought to have a strong impact on sequencing performance had surprisingly little effect. Thus, primers with high or low percent G + C or Tm, strong secondary priming scores or long 3' homopolymeric stretches yielded excellent sequences with the dRhodamine dye terminator reagents, although these characteristics had a stronger effect when the old rhodamine reagents were used. The old rhodamine reagents gave sequences with a similar average read length, but the number of errors and ambiguities or "N's" was consistently higher. Moreover, the effects of the primer physico-chemical characteristics were also more evident with the old rhodamine dyes. We conclude that under optimal sequencing conditions with highly pure template and primer, many of the commonly applied primer design parameters are dispensable, particularly when using one of the new generation of sequencing reagents such as the dichlororhodamine dye terminators.
Assuntos
Primers do DNA/síntese química , Desenho de Fármacos , Análise de Sequência de DNA , Autoanálise , Fenômenos Químicos , Físico-Química , Primers do DNA/química , Dimerização , Corantes Fluorescentes , Indicadores e Reagentes , Rodaminas , SoftwareRESUMO
The NPM-MLF1 fusion protein is expressed in blasts from patients with myelodysplasia/acute myeloid leukemia (MDS/AML) containing the t(3;5) chromosomal rearrangement. Nucleophosmin (NPM), a previously characterized nucleolar phosphoprotein, contributes to two other fusion proteins found in lympho-hematopoietic malignancies, anaplastic large cell lymphoma (NPM-ALK) and acute promyelocytic leukemia (NPM-RARalpha). By contrast, the function of the carboxy-terminal fusion partner, myelodysplasia/myeloid leukemia factor 1 (MLF1), is unknown. To aid in understanding normal MLF1 function, we isolated the murine cDNA, determined the chromosomal localization of Mlf1, and defined its tissue expression by in situ hybridization. Mlf1 was highly similar to its human homologue (86% and 84% identical nucleotide and amino acid sequence, respectively) and mapped to the central region of chromosome 3, within a segment lacking known mouse mutations. Mlf1 tissue distribution was restricted during both development and postnatal life, with high levels present only in skeletal, cardiac, and selected smooth muscle, gonadal tissues, and rare epithelial tissues including the nasal mucosa and the ependyma/choroid plexus in the brain. Mlf1 transcripts were undetectable in the lympho-hematopoietic organs of both the embryonic and adult mouse, suggesting that NPM-MLF1 contributes to the genesis of MDS/AML in part by enforcing the ectopic overexpression of MLF1 within hematopoietic tissues.
Assuntos
Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Leucemia Mieloide/genética , Síndromes Mielodisplásicas/genética , Proteínas/genética , Doença Aguda , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas/metabolismo , Homologia de Sequência , Distribuição Tecidual/fisiologiaRESUMO
Mucosa-associated lymphoid tissue (MALT) lymphomas most frequently involve the gastrointestinal tract and are the most common subset of extranodal non-Hodgkin lymphoma (NHL). Here we describe overexpression of BCL10, a novel apoptotic signalling gene that encodes an amino-terminal caspase recruitment domain (CARD), in MALT lymphomas due to the recurrent t(1;14)(p22;q32). BCL10 cDNAs from t(1;14)-positive MALT tumours contained a variety of mutations, most resulting in truncations either in or carboxy terminal to the CARD. Wild-type BCL10 activated NF-kappaB but induced apoptosis of MCF7 and 293 cells. CARD-truncation mutants were unable to induce cell death or activate NF-kappaB, whereas mutants with C-terminal truncations retained NF-kappaB activation but did not induce apoptosis. Mutant BCL10 overexpression might have a twofold lymphomagenic effect: loss of BCL10 pro-apoptosis may confer a survival advantage to MALT B-cells, and constitutive NF-kappaB activation may provide both anti-apoptotic and proliferative signals mediated via its transcriptional targets.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Caspases/metabolismo , Linfoma de Zona Marginal Tipo Células B/genética , Proteínas de Neoplasias/genética , Sequência de Aminoácidos , Proteína 10 de Linfoma CCL de Células B , Sítios de Ligação , Northern Blotting , Morte Celular/genética , Linhagem Celular , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 14/genética , DNA/química , DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Masculino , Dados de Sequência Molecular , Mutação , NF-kappa B/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Translocação Genética , Células Tumorais CultivadasRESUMO
Several recent studies have examined the possibility of producing tumor-specific cytotoxicity with various enzyme/ prodrug combinations. The enzymes are targeted to tumor cells either with antibodies (ADEPT, antibody directed enzyme prodrug therapy) or with viruses (VDEPT). The goal of the present study was to identify an appropriate enzyme for use in activating the prodrug 7-ethyl-10-[4-(1-piper-idino)-1-piperidino]carbonyloxycamptothe cin (CPT-11). In this study, we compared the efficiency of CPT-11 metabolism by rabbit and human carboxylesterases in in vitro and in situ assays. Although the rabbit and human enzymes are very similar (81% identical; 86% homologous) and the active site amino acids are 100% identical, the rabbit enzyme was 100-1000-fold more efficient at converting CPT-11 to SN-38 in vitro and was 12-55-fold more efficient in sensitizing transfected cells to CPT-11. In vivo, Rh30 rhabdomyosarcoma cells expressing the rabbit carboxylesterase and grown as xenografts in immune-deprived mice were also more sensitive to CPT-11 than were control xenografts or xenografts expressing the human enzyme. Each of the three types of xenografts regressed when the mice were treated with CPT-11 given i.v. at 2.5 mg of CPT-11/kg/daily for 5 days/week for 2 weeks [(dx5)2] (one cycle of therapy), repeated every 21 days for a total of three cycles. However, following cessation of treatment, recurrent tumors were detected in seven of seven mice bearing control Rh30 xenografts and in two of seven mice bearing Rh30 xenografts that expressed the human enzyme. No tumors recurred in mice bearing xenografts that expressed the rabbit carboxylesterase. We conclude that rabbit carboxylesterase/CPT-11 may be a useful enzyme/prodrug combination.
Assuntos
Antineoplásicos Fitogênicos/metabolismo , Camptotecina/análogos & derivados , Hidrolases de Éster Carboxílico/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Pró-Fármacos/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Biotransformação , Camptotecina/metabolismo , Camptotecina/uso terapêutico , Carboxilesterase , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/uso terapêutico , Catálise , Divisão Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Irinotecano , Camundongos , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Transplante de Neoplasias , Neoplasias Experimentais/patologia , Nitrobenzenos/metabolismo , Fenilacetatos/metabolismo , Pró-Fármacos/uso terapêutico , Coelhos , Células Tumorais CultivadasRESUMO
The AML1/core binding factor beta (CBFbeta) transcription factor is essential for definitive hematopoiesis; however, the downstream pathways through which it functions remain incompletely defined. Using a differential cloning approach to define components of this pathway, we have identified a novel gene designated HERF1 (for hematopoietic RING finger 1), whose expression during development is dependent on the presence of functional AML1/CBFbeta. HERF1 contains a tripartite RING finger-B box-alpha-helical coiled-coil domain and a C-terminal region homologous to the ret proto-oncogene-encoded finger protein. Expression of HERF1 during embryogenesis coincides with the appearance of definitive erythropoiesis and in adult mice is restricted to erythroid cells, increasing 30-fold during terminal differentiation. Importantly, inhibition of HERF1 expression blocked terminal erythroid differentiation of the murine erythroleukemia cell line MEL, whereas its overexpression induced erythroid maturation. These results suggest an important role for this protein in erythropoiesis.
Assuntos
Proteínas de Transporte/genética , Diferenciação Celular/genética , Eritropoese/genética , Proteínas Proto-Oncogênicas , Dedos de Zinco/genética , Animais , Linhagem Celular , Clonagem Molecular , Subunidade alfa 2 de Fator de Ligação ao Core , Proteínas de Ligação a DNA , Dimetil Sulfóxido , Regulação da Expressão Gênica no Desenvolvimento/genética , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , RNA Mensageiro/genética , Tetraciclina/farmacologia , Fatores de Transcrição/genética , Proteínas com Motivo TripartidoRESUMO
The murine transcription factor murine cyclin D-binding Myb-like protein (mDmp1) arrests the cell cycle in G1 phase, through an activity that can be overridden by direct interaction with the D-type cyclins. Here, we describe the identification, sequence, chromosomal localization, and expression of the human cognate, hDMP1. The hDMP1 cDNA contains a 2280bp open reading frame that shares a high degree of identity with the mDmp1 coding region. The 4.4kb hDMP1 messenger RNA is ubiquitously expressed in normal human tissues, with highest levels in testis and substructures within the brain. By use of fluorescence in situ hybridization with a human genomic P1 probe, we assigned hDMP1 to chromosome 7, band q21. This chromosomal region is frequently deleted as part of the 7q-minus and monosomy 7 abnormalities of human acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We analyzed hDMP1 copy number by fluorescence in situ hybridization in leukemic blasts from nine patients with abnormalities of the long arm of chromosome 7, and in each case one allele of the hDMP1 gene was deleted. Functional analysis of the mDmp1 protein has shown that it negatively regulates cell proliferation, which suggests that this gene is a candidate suppressor of malignant transformation. Further study will be needed to determine whether gene-specific mutations implicate hDMP1 as a tumor suppressor in acute leukemias with deletions of the long arm of chromosome 7 or in other types of human malignancy.
Assuntos
Cromossomos Humanos Par 7/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Ciclo Celular/genética , Divisão Celular/genética , Mapeamento Cromossômico , Clonagem Molecular , Ciclina D , Ciclinas/metabolismo , Deleção de Genes , Genes Supressores de Tumor , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Leucemia/genética , Dados de Sequência Molecular , Ligação Proteica/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) chromosomal translocation, is thought to drive the leukemic transformation of early B-cell precursors by repressing an evolutionarily conserved apoptotic pathway. To test this hypothesis, we sought to identify downstream targets of E2A-HLF in t(17;19)+ pro-B leukemia cells (UOC-B1) that had been transfected with a zinc-inducible vector encoding a dominant-negative suppressor (E2A-HLF[dn]) of the oncoprotein. Representational difference analysis of mRNAs from E2A-HLF(dn)+ UOC-B1 cells grown with (E2A-HLF inactive) or without (E2A-HLF active) the addition of zinc yielded several differentially expressed cDNA fragments that were individually subcloned. Two of the clones, designated F-5 and G-4, hybridized with mRNAs that were upregulated by E2A-HLF. Levels of both transcripts declined sharply within 8 to 12 hours after suppression of E2A-HLF DNA-binding activity, becoming undetectable after 96 hours. The F-5 cDNA was identified as a portion of ANNEXIN VIII, whose product was expressed in promyelocytic leukemia cells and UOC-B1 cells, but not in other leukemic cell lines. A novel full-length cDNA cloned with the G-4 fragment encoded a protein that we have named SRPUL (sushi-repeat protein upregulated in leukemia). It is normally expressed in heart, ovary, and placenta, but could not be detected in leukemic cell lines other than UOC-B1. Neither protein prevented apoptosis in interleukin-3-dependent murine pro-B cells, suggesting that they have paraneoplastic roles in leukemias that express E2A-HLF, perhaps in the disseminated intravascular coagulopathy and hypercalcemia that characterize these cases.
Assuntos
Proteínas de Ligação a DNA/genética , Genes Neoplásicos , Proteínas de Membrana/genética , Proteínas de Neoplasias , Proteínas de Fusão Oncogênica/genética , Sequência de Aminoácidos , Anexinas/genética , Apoptose , Linhagem Celular , Clonagem Molecular , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Dados de Sequência Molecular , Sequências Repetitivas de Aminoácidos , Selectinas/química , Selectinas/genética , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
We have isolated a cDNA encoding a rabbit carboxylesterase (CE; EC 3.1.1.1) that converts the camptothecin-derived prodrug irinotecan (CPT-11) to the potent topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothecin. NH2-terminal amino acid sequencing of a purified rabbit CE allowed the design of redundant oligonucleotides to perform PCR from rabbit liver cDNA. DNA sequencing of the PCR product confirmed the identity of the clone, and after both 5' and 3' rapid amplification of cDNA ends, oligonucleotide primers were designed to amplify the entire cDNA. The 1698-bp open reading frame encoded a 565-amino acid protein containing the characteristic CE B-1 and B-2 motifs, a hydrophobic NH2-terminal leader sequence, and the COOH-terminal residues HIEL that are thought to be responsible for protein localization in the endoplasmic reticulum. Transient expression of the cDNA in COS-7 cells resulted in CE activity in cell extracts and increased the sensitivity of cells to CPT-11. Additionally, stable expression of the rabbit liver CE cDNA in the human glioma U-373 MG cell line resulted in a 56-fold decrease in the IC50 value for CPT-11, whereas the expression of a human alveolar macrophage cDNA encoding a highly homologous CE produced no change in drug sensitivity.
Assuntos
Antineoplásicos Fitogênicos/metabolismo , Camptotecina/análogos & derivados , Hidrolases de Éster Carboxílico/genética , DNA Complementar/isolamento & purificação , Pró-Fármacos/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Sequência de Bases , Células COS/efeitos dos fármacos , Células COS/enzimologia , Camptotecina/química , Camptotecina/metabolismo , Camptotecina/farmacologia , Carboxilesterase , Hidrolases de Éster Carboxílico/isolamento & purificação , Hidrolases de Éster Carboxílico/metabolismo , Sobrevivência Celular/efeitos dos fármacos , DNA Complementar/química , Glioma/enzimologia , Humanos , Irinotecano , Fígado/enzimologia , Dados de Sequência Molecular , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Coelhos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Rhombotin-2 (RBTN-2) is a proto-oncogene only in the context of T lymphocytes. We postulated that the oncogenic effect of RBTN-2 in T cells is likely mediated by binding protein(s) with T cell-specific expression. By screening a T cell cDNA library, we identified a novel ets transcription factor that binds RBTN-2. This protein was named elf-2 because its DNA-binding domain is virtually identical to that of ets family member elf-1. Northern analyses showed similar levels of two elf-2 transcripts (3.5 kb and 3.8 kb) in all tissues except thymus. Thymocytes expressed four- to 10-fold greater amounts of the 3.5 kb transcript than other tissues. Sequence analyses of cDNA clones indicated that these transcripts encode proteins differing only at their amino termini, and likely represent alternatively spliced isoforms. These isoforms (elf-2a and elf-2b) contain identical RBTN-2 binding regions and DNA-binding domains. Elf-2b lacks a putative transactivation domain. The expression patterns suggest that RBTN-2 normally interacts equally with elf-2a and elf-2b. In contrast, when RBTN-2 is inappropriately expressed in T cells, RBTN-2 would interact predominantly with elf-2b; this interaction may lead to T cell proliferation.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Leucemia de Células T/metabolismo , Metaloproteínas/metabolismo , Linfócitos T/química , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Pré-Escolar , Proteínas de Ligação a DNA/isolamento & purificação , Células HL-60 , Humanos , Proteínas com Domínio LIM , Camundongos , Dados de Sequência Molecular , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas , Análise de Sequência de DNA , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , LevedurasRESUMO
The purity of 208 crude synthetic 25- and 50-base oligonucleotides synthesized in 71 DNA core facilities was assessed by capillary electrophoresis (CE), and the average coupling efficiency of each synthesis was determined. The median average coupling efficiencies of the 25-mers and 50-mers were 98.9% and 98.7%, respectively, and 85% of the samples exceeded the minimum industry standard of 98% average coupling efficiency. The overall yields estimated by on-line trityl monitors showed poor agreement with the empirically determined yield, and accuracy of the monitors decreased as synthesis efficiency decreased. The performance of the unpurified 25-base oligonucleotides, ranging in purity from 14% to 94%, as primers for automated DNA sequencing was evaluated. Over 85% of these oligonucleotides exhibited an unedited sequencing accuracy of > 97.5% over the 400-base test sequence. Surprisingly, sequencing performance was not strictly related to primer purity, though a marked loss of performance was observed for primers < or = 70% pure (< or = 98.5% coupling efficiency). Thus, the vast majority of the oligonucleotides synthesized by the 71 core facilities participating in this study were of high quality and performed well as sequencing primers without post-synthesis purification or desalting. Finally, our results suggest that an increase in the standard minimum performance specifications of DNA synthesis instruments and reagents from > or = 98% to > or = 98.5% average coupling efficiency, or the development of rapid, inexpensive and efficient methods to detect syntheses below the 98.5% threshold, could obviate post synthesis purification of sequencing primers.
Assuntos
Primers do DNA , Oligonucleotídeos/metabolismo , Análise de Sequência de DNARESUMO
A fusion gene between nucleophosmin (NPM) and myelodysplasia/myeloid leukemia factor 1 (MLF1) is formed by a recurrent t(3;5)(q25.1;q34) in myelodysplastic syndrome and acute myeloid leukemia. Here we report the identification of a novel gene, MLF2, which contains an open reading frame of 744 bp encoding a 248-amino-acid protein highly related to the previously identified MLF1 protein (63% similarity, 40% identity). In contrast to the tissue-restricted expression pattern of MLF1, the MLF2 messenger RNA is expressed ubiquitously. The MLF2 gene locus was mapped by fluorescence in situ hybridization to human chromosome 12p13, a chromosomal region frequently involved in translocations and deletions in acute leukemias of lymphoid or myeloid lineage. In a physical map of chromosome 12, MLF2 was found to reside on the yeast artificial chromosome clone 765b9. Southern blotting analysis of malignant cell DNAs prepared from a series of acute lymphoblastic leukemia cases with translocations involving chromosome arm 12p, as well as a group of acute myeloid leukemias with various cytogenetic abnormalities, failed to reveal MLF2 gene rearrangements.
Assuntos
Cromossomos Humanos Par 12 , Cromossomos Humanos Par 3 , Cromossomos Humanos Par 5 , Expressão Gênica , Translocação Genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Ciclo Celular , Aberrações Cromossômicas , Transtornos Cromossômicos , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Proteínas de Ligação a DNA , Humanos , Hibridização in Situ Fluorescente , Leucemia Mieloide/genética , Dados de Sequência Molecular , Família Multigênica , Neoplasias/genética , Proteínas Nucleares/genética , Nucleofosmina , Fases de Leitura Aberta , Especificidade de Órgãos , Biossíntese de Proteínas , Proteínas/química , RNA Mensageiro/biossíntese , Homologia de Sequência de AminoácidosRESUMO
A double-stranded (ds)DNA template of "unknown" sequence was distributed to approximately 80 core DNA sequencing laboratories by the Association of Biomolecular Resource Facilities (ABRF) for automated DNA sequence analysis. Forty-four different facilities responded with 83 usable sequence submissions. These sequences were grouped by both sequencing protocol (dye-primer or dye-terminator) and whether manually edited or not. The sequences were aligned with the known sequence, and the number of correct base calls, insertions, deletions, no-calls and miscalls were determined for each group. The dye-primer sequencing protocol provided the longest and most accurate sequence. The edited dye-primer data were > 95% accurate out to 400-450 bp, while the edited dye-terminator data could call only 300-350 bases at this accuracy. However, 75% of the laboratories in this sampling preferred the dye-terminator protocol, presumably because of its versatility and convenience. Laboratories that manually edited the automatically called data were able to obtain an additional 100 bases of good sequence when the dye-primer protocol was used. Surprisingly though, editing of dye-terminator results did not increase the amount of good sequence, although the dye-terminator protocol had a superior base-calling ability within the first 100 bases of called sequence.
Assuntos
Autoanálise/estatística & dados numéricos , Laboratórios/estatística & dados numéricos , Análise de Sequência de DNA/métodos , Composição de Bases , Corantes , DNA/química , Primers do DNA , Sensibilidade e Especificidade , Análise de Sequência de DNA/estatística & dados numéricos , Moldes GenéticosRESUMO
Growth of influenza viruses in embryonated eggs frequently results in the selection of virus variants with amino acid changes near the receptor-binding pocket of the hemagglutinin molecule, yet the mechanism by which this third form of influenza variation occurs (the other two being antigenic drift and shift) has not been clearly defined. Because egg-mediated variation might affect influenza vaccine and surveillance programs, we have initiated studies to determine the site(s) of variant virus selection within the embryonated egg. In this report we show that both the cells of the chorioallantoic membrane (CAM) and the fluids from embryonated chicken eggs are capable of selecting variant influenza viruses, but that these variants are distinct at the molecular level depending on the conditions of virus propagation. Serial passage of viruses in cells of the chorioallantoic membrane selects one set of variants which possess specific amino acid changes near the receptor binding pocket of the hemagglutinin molecule characteristic of viruses grown in embryonated eggs. However, passage of the same viruses in mammalian tissue culture cells supplemented with egg fluids selects a separate set of hemagglutinin variants also characteristic of viruses grown in eggs, yet at different residues from those observed following passage in CAM. These results suggest that two separate mechanisms may exist in the embryonated egg that lead to the selection of variant influenza viruses: one at the cellular level and another at the extracellular level.
Assuntos
Variação Genética , Hemaglutininas Virais/química , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza A/genética , Estrutura Secundária de Proteína , Replicação Viral , Alantoide , Sequência de Aminoácidos , Animais , Linhagem Celular , Embrião de Galinha , Galinhas , Córion , Cães , Ovos , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas Virais/genética , Vírus da Influenza A/classificação , Vírus da Influenza A/fisiologia , Proteínas do Envelope Viral/químicaRESUMO
Alveolar rhabdomyosarcoma, a malignant tumor of skeletal muscle, is characterized by a chromosomal translocation, t(2;13)(q35;q14). This translocation is associated with a structural rearrangement of the gene encoding PAX3, a presumed transcriptional regulator expressed exclusively during embryogenesis. The breakpoint results in a fusion between PAX3 and a gene provisionally named ALV, a novel member of the forkhead family of transcription factors. In PAX3-ALV, the structural integrity of both PAX3 DNA-binding regions, the paired box and homeodomain, are retained while the putative transcriptional activation domain of PAX3 is replaced by the bisected forkhead DNA-binding domain of ALV. Formation of chimeric transcription factors has now been implicated in diverse human tumors of myogenic, hematopoietic, neuroectodermal, and adipocytic origin, suggesting that transcriptional deregulation is a common mechanism of tumorigenesis.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/metabolismo , Fatores de Transcrição/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 2 , Clonagem Molecular , Sequência Conservada , Humanos , Dados de Sequência Molecular , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Translocação Genética , Células Tumorais CultivadasRESUMO
The possibility that linear epitopes on the haemagglutinin-neuraminidase (HN) surface glycoprotein of human parainfluenza virus type 3 (PIV-3) might induce neutralizing antibodies after virus infection was investigated. Thirty-seven peptides, representing 64% of the extramembranous portion of the HN molecule of PIV-3, were synthesized. Their ability to bind to 14 neutralizing murine monoclonal antibodies (mAbs) specific for HN or 26 high-titre human serum samples were tested in a direct enzyme-linked immunosorbent assay (ELISA) and in an indirect competition ELISA. None of the synthetic peptides reacted with any of the mAbs or serum samples in the direct test and none of 11 synthetic peptides tested blocked mAbs from binding to HN in the competition ELISA. These findings suggest that synthetic peptides cannot be used to imitate the known neutralizing epitopes on the HN. Analyses of reduced and non-reduced HN in ELISA and immunoblot assays confirmed that protein folding and tertiary structure are essential for epitope formation in these neutralizing sites. However, some children's sera analysed by immunoblotting contained antibodies to an uncharacterized linear epitope(s) not recognized by our panel of mAbs, raising the possibility that a neutralizing linear epitope does exist on the HN of PIV-3.
Assuntos
Antígenos Virais/química , Proteína HN/imunologia , Vírus da Parainfluenza 3 Humana/imunologia , Peptídeos/imunologia , Vacinas Sintéticas/imunologia , Sequência de Aminoácidos , Animais , Western Blotting , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Proteína HN/química , Haplorrinos , Humanos , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The covalent attachment of fatty acid moieties to proteins is a widespread post-translational modification of viral and cell proteins yet the functional consequences of acylation are not well understood. We have determined that the A/Japan/305/57 influenza virus hemagglutinin (HA) contains three potential acylation sites at cysteine residues 211, 218 and 221 in the cytoplasmic domain of the molecule. Site-directed mutagenesis of one or more of these sites has no effect on biosynthesis, transport or receptor binding activity of the molecule; however, modification of any single site is sufficient to abolish completely or inhibit severely membrane fusion activity, a function essential for virus infectivity. We present a molecular model of the transmembrane and cytoplasmic domains of the HA to illustrate the potential orientation of these fatty acids and to provide a conceptual framework for further experimentation.
Assuntos
Ácidos Graxos/metabolismo , Hemaglutininas Virais/fisiologia , Vírus da Influenza A/fisiologia , Fusão de Membrana , Acilação , Sequência de Aminoácidos , Animais , Linhagem Celular , Gráficos por Computador , Imunofluorescência , Genes Virais , Vetores Genéticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Hemaglutininas Virais/genética , Vírus da Influenza A/genética , Bicamadas Lipídicas , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ácido Palmítico , Ácidos Palmíticos/metabolismo , Conformação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Antigenic variants of influenza virus A/Mem/1/71-Bel/42 (H3N1) selected with monoclonal antibodies and having single substitutions in their hemagglutinins were examined for their ability to hemagglutinate and hemolyse erythrocytes coated with different gangliosides. The majority of variants, including one with a substitution near the receptor-binding site (Asn-133----Lys), did not differ from the parent in specificity for receptor molecules. However, a substitution in HA1 at residue 205 (Ser----Tyr), which is distant from the receptor-binding site in antigenic site D, affected hemagglutination and hemolysis of erythrocytes coated with sialyl-paraglobosides. The variant preferentially recognized N-acetylneuraminic acid-alpha 2,6-galactose linkages to sialylparaglobosides, whereas the parent and other variants preferentially recognized N-acetylneuraminic acid-alpha 2,3-galactose linkages. In the trimeric hemagglutinin molecule, residue 205 is located across the subunit interface from the receptor-binding site. The bulky hydrophobic tyrosine in the variant may cause a conformational change in the receptor-binding pocket on the neighboring subunit and influence receptor binding.