RESUMO
QBP359 is an IgG1 human monoclonal antibody that binds with high affinity to human CCL21, a chemokine hypothesized to play a role in inflammatory disease conditions through activation of resident CCR7-expressing fibroblasts/myofibroblasts. The pharmacokinetics (PK) and pharmacodynamics (PD) of QBP359 in non-human primates were characterized through an integrated approach, combining PK, PD, immunogenicity, immunohistochemistry (IHC) and tissue profiling data from single- and multiple-dose experiments in cynomolgus monkeys. When compared with regular immunoglobulin typical kinetics, faster drug clearance was observed in serum following intravenous administration of 10 mg/kg and 50 mg/kg of QBP359. We have shown by means of PK/PD modeling that clearance of mAb-ligand complex is the most likely explanation for the rapid clearance of QBP359 in cynomolgus monkey. IHC and liquid chromatography mass spectrometry data suggested a high turnover and synthesis rate of CCL21 in tissues. Although lymphoid tissue was expected to accumulate drug due to the high levels of CCL21 present, bioavailability following subcutaneous administration in monkeys was 52%. In human disease states, where CCL21 expression is believed to be expressed at 10-fold higher concentrations compared with cynomolgus monkeys, the PK/PD model of QBP359 and its binding to CCL21 suggested that very large doses requiring frequent administration of mAb would be required to maintain suppression of CCL21 in the clinical setting. This highlights the difficulty in targeting soluble proteins with high synthesis rates.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Quimiocina CCL21/antagonistas & inibidores , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Animais , Afinidade de Anticorpos , Cromatografia Líquida , Humanos , Imuno-Histoquímica , Macaca fascicularis , Espectrometria de MassasRESUMO
The migration of neutrophils into sites of acute and chronic inflammation is mediated by chemokines. We used degenerate-primer reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze chemokine receptor expression in neutrophils and identify novel receptors. RNA was isolated from human peripheral blood neutrophils and from neutrophils that had been stimulated for 5 h with granulocyte-macrophage colony-stimulating factor or by coculturing with primary human bronchial epithelial cells. Amplification products were cloned, and clone redundancy was determined. Seven known G-protein-coupled receptors were identified among 38 clones-CCR1, CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1-as well as a novel gene, EX33. The full-length EX33 clone was obtained, and an in silico approach was used to identify the putative murine homologue. The EX33 gene encodes a 396-amino-acid protein with limited sequence identity to known receptors. Expression studies of several known chemokine receptors and EX33 revealed that resting neutrophils expressed higher levels of CXCRs and EX33 compared with activated neutrophils. Northern blot experiments revealed that EX33 is expressed mainly in bone marrow, lung, and peripheral blood leukocytes. Using RT-PCR analysis, we showed more abundant expression of EX33 in neutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression among leukocytes.
Assuntos
Quimiotaxia de Leucócito/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Neutrófilos/metabolismo , Receptores de Superfície Celular/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Western Blotting , Clonagem Molecular , DNA Complementar/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Genes , Humanos , Dados de Sequência Molecular , Neutrófilos/efeitos dos fármacos , Especificidade de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Receptores de Superfície Celular/biossíntese , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/genética , Receptores Acoplados a Proteínas G , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Epithelial cells lining the airways are thought to play a prominent role in respiratory diseases. We utilized cDNA representational difference analysis to identify the genes in which expression is induced by the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta in primary human bronchial epithelial cells and hence are relevant to airway inflammation. Hybridization of the subtraction product to arrayed cDNAs indicated that known tumor necrosis factor-alpha- and interleukin-1beta-inducible genes such as B94, Zfp36, and regulated on activation normal T cell expressed and secreted were represented, confirming the success of the subtraction experiment. A 1,152-clone library potentially representing genes with higher transcript levels in cytokine-treated human bronchial epithelial cells was generated and sequenced. Sequence similarity searches indicated that these clones represented 57 genes of known function, 1 gene of unknown function, 6 expressed sequence tags, and 2 novel sequences. The expression of 19 of these clones was studied by a combination of Northern blotting and RT-PCR analyses and confirmation of differential expression for 10 known genes, 2 expressed sequence tags, and a novel sequence not represented in any of the public databases was obtained. Thus cDNA representational difference analysis was utilized to isolate known and novel differentially expressed genes, which putatively play a role in airway inflammation.
Assuntos
Citocinas/metabolismo , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica/métodos , Mediadores da Inflamação/metabolismo , Mucosa Respiratória/metabolismo , Northern Blotting , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Células Cultivadas , Citocinas/farmacologia , DNA Complementar/genética , DNA Complementar/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Etiquetas de Sequências Expressas , Humanos , Mediadores da Inflamação/farmacologia , Interleucina-1/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Pneumonia/genética , Pneumonia/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Neutrophils are the first cell type to migrate out of the vascular space and into the inflammatory site during an acute inflammation. However, in chronic inflammatory diseases, such as chronic obstructive pulmonary disease (COPD), a lack of clearance of neutrophils, imbalance between inflammatory mediators produced by neutrophils and their natural inhibitors make these cells a potential cause of tissue destruction in lung disease. Neutrophilic inflammation is generally characterised by high levels of local expression of activating cytokines (e.g., GM-CSF). Only a few studies have been published so far that have investigated the expression of genes preferentially expressed in activated neutrophils. The isolation of such genes, however, can lead to a better understanding of inflammatory disease and the identification of potential novel therapeutic targets or markers of the disease. We performed representational difference analysis of cDNA, a sensitive PCR-based subtractive enrichment procedure, and isolated 12 genes, 1 EST clone, and 3 sequences not represented in the public databases. Differential expression for 9 of these clones was confirmed by Northern hybridisation. Of the above nine transcripts three were chosen and shown to be up-regulated in neutrophils cocultured with stimulated primary human bronchial epithelial cells using a semiquantitative RT-PCR approach. Among the known genes identified were HM-74, CIS1, Cathepsin C, alpha-enolase, CD44, and the gene Translocation Three Four (TTF), most of them previously not known to be involved in GM-CSF induced neutrophil activation. Along with its tissue and cellular distribution we also derived the complete cDNA sequence and genomic structure of CIS1 using an in silico approach. In addition, we also report the initial characterisation of a novel gene, P1-89 that is primarily expressed in granulocytes and is up-regulated in activated cells. Our results identify several important genes associated with neutrophil activation and can lead to a better understanding of the molecular mechanisms of neutrophilic inflammations.
Assuntos
DNA Complementar/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Neutrófilos/metabolismo , Proteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Brônquios/metabolismo , Catepsina C/genética , Células Cultivadas , Técnicas de Cocultura , Células Epiteliais/metabolismo , Etiquetas de Sequências Expressas , Biblioteca Gênica , Granulócitos/metabolismo , Humanos , Receptores de Hialuronatos/genética , Proteínas Imediatamente Precoces/genética , Inflamação , Modelos Genéticos , Dados de Sequência Molecular , Fosfopiruvato Hidratase/biossíntese , Fosfopiruvato Hidratase/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Proteínas Supressoras da Sinalização de Citocina , Distribuição Tecidual , Fatores de Transcrição , Regulação para Cima , Proteínas rho de Ligação ao GTPRESUMO
The identification of genetic factors important in lung development and function will help in understanding the underlying molecular mechanisms of respiratory disease. Representational difference analysis of cDNA (cDNA-RDA) is a PCR-based subtractive enrichment procedure for the isolation of differentially expressed genes. We performed cDNA-RDA and isolated genes expressed more abundantly in fetal and adult lungs. Fifty-four clones potentially representing genes with higher transcript levels in the fetal lung were sequenced. Sequence similarity searches indicated that these clones included 12 known genes, a discoidin-like domain-containing gene, six expressed sequence tags (ESTs), and one novel sequence. Fifty-six clones potentially representing genes expressed more abundantly in the adult lung were also cloned and sequenced. Of these, 16 known human genes were represented along with two sequences significantly similar to known mouse genes and two novel sequences. Several of these known genes are implicated in stress response and lung protection. Thus cDNA-RDA was successfully used to isolate known and novel differentially expressed genes, which putatively play an important role in human lung development.
Assuntos
Envelhecimento/fisiologia , DNA Complementar/genética , Feto/fisiologia , Expressão Gênica , Pulmão/fisiologia , Adolescente , Adulto , Sequência de Aminoácidos/genética , Animais , Animais Recém-Nascidos/metabolismo , Sequência de Bases/genética , Água Corporal/metabolismo , Feminino , Humanos , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Masculino , Dados de Sequência Molecular , RNA Mensageiro/genética , Estresse Fisiológico/genética , Técnica de SubtraçãoRESUMO
We have cloned a gene (pepF) encoding a serine carboxypeptidase, proteinase F (PEPF), from Aspergillus niger. The sequences were identified in a phage lambda genomic DNA library using a synthetic probe based on the N-terminal sequence of PEPF. Nucleotide sequence data from pepF genomic and cDNA clones reveals that it is composed of four exons of 199, 283, 227 and 881 bp, interrupted by three introns of 53, 69 and 59 bp. The sequence of pepF codes for a polypeptide of 530 amino acids (aa), of which the first 52 aa are not present in the mature PEPF. This region may represent a prepro sequence that is removed by proteolytic cleavage as a monobasic cleavage site (Lys52). Northern blot analysis of total cellular RNA extracted from A. niger cells indicates that pepF is transcribed as a single 1.8-kb mRNA, which is regulated by nitrogen and carbon repression, specific induction and the pH of the culture medium.
Assuntos
Aspergillus niger/enzimologia , Aspergillus niger/genética , Genes Fúngicos , Metaloendopeptidases/genética , Serina Endopeptidases/genética , Sequência de Aminoácidos , Proteínas de Bactérias , Bacteriófago lambda , Sequência de Bases , Northern Blotting , Clonagem Molecular , Expressão Gênica , Biblioteca Gênica , Concentração de Íons de Hidrogênio , Íntrons , Metaloendopeptidases/biossíntese , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Fases de Leitura Aberta , Plantas/genética , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Aspergillus niger secretes a number of enzymes, including proteases, into its culture fluid. The regulation of the two major acidic extracellular proteases, pepA and pepB, was investigated using Northern analyses. Our data suggest that the regulation of pepA and pepB expression occurs predominantly at the level of mRNA content and that, while they are regulated in a similar manner, differences are also clear in their expression. Both genes were found to be under complex regulatory control. The expression of the two genes could be turned off by the presence of good nitrogen or carbon sources in the media, and external protein sources did not induce expression of either gene under conditions of carbon and nitrogen repression. The pH of the medium also played a major role in their regulation as the expression of both genes was completely turned off under alkaline conditions, even when grown in media lacking good nitrogen and carbon sources but containing proteins. We isolated clones containing 5' non-coding sequences of the pepA gene from a lambda genomic library with a pepA specific probe. Analysis and comparison of the promoter sequences of the pepA and pepB genes revealed that both contain several putative AREA- and CREA-binding sites and they also share an 18-bp-long sequence which is 83% identical in these two genes.
Assuntos
Ácido Aspártico Endopeptidases/genética , Aspergillus niger/enzimologia , Aspergillus niger/genética , Sequência de Bases , Carbono/metabolismo , DNA , Sondas de DNA , DNA Fúngico , Endopeptidases , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Nitrogênio/metabolismo , Regiões Promotoras Genéticas , RNA , RNA Fúngico , RNA Mensageiro , Análise de Sequência de DNARESUMO
We have cloned the pepE gene of Aspergillus niger, encoding an aspartic protease (PEPE), by screening a lambda genomic DNA library with a heterologous probe, the Neurospora crassa gene coding for a vacuolar proteinase. Sequencing of pepE genomic and cDNA clones revealed that the gene contains three introns, which are 91, 56 and 58-bp long. The deduced protein consists of 398 amino acids, has a putative signal sequence to allow transport into the endoplasmic reticulum and probably undergoes a second proteolytic processing step at its N terminus to yield the mature enzyme. The putative mature part of PEPE has extensive homology with other aspartic proteinases such as pepsins, cathepsins and, in particular, with proteinase A of Saccharomyces cerevisiae and pepsin 1 of Candida albicans. Northern blot analyses revealed that cells contain an abundant pepE transcript whose amount does not change upon carbon or nitrogen limitation, the presence of proteins in the medium or changes in the pH of the medium. We also show that pepC, the A. niger homologue of yeast protease B, is also expressed constitutively under these conditions.
Assuntos
Ácido Aspártico Endopeptidases/genética , Aspergillus niger/genética , Proteínas Fúngicas , Genes Fúngicos/genética , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/biossíntese , Aspergillus niger/enzimologia , Sequência de Bases , Southern Blotting , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Íntrons/genética , Dados de Sequência Molecular , RNA Mensageiro/genética , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Transcrição GênicaRESUMO
Serine proteases constitute an important group of extra- and intracellular proteases in fungi. These enzymes are characterized by conserved regions around the active site residues, Asp, His and Ser. Based on this amino acid (aa) sequence conservation, we have used degenerate primer PCR to isolate subtilisin-specific genomic probes from Aspergillus niger, and cloned a gene, pepD, by screening a lambda genomic library using a PCR probe. The pepD gene contains three putative introns, which are 51-, 47- and 55-bp long and has an open reading frame coding for a protein which consists of 416 aa. The deduced aa sequence shows similarity to subtilisin-like proteases, in particular to fungal alkaline proteases. Signal sequence cleavage prediction indicates that the first 20 aa are probably removed upon transfer to the endoplasmic reticulum. The conservation of the pro-enzyme cleavage site in fungal alkaline proteases suggests that the mature protein is derived from this polypeptide via the removal of an additional 101 aa, resulting in a mature 30,294-Da enzyme consisting of 295 aa.
Assuntos
Aspergillus niger/enzimologia , Aspergillus niger/genética , Endopeptidases/genética , Genes Fúngicos , Subtilisinas/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Primers do DNA , Endopeptidases/biossíntese , Éxons , Biblioteca Genômica , Íntrons , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Sinais Direcionadores de Proteínas/genética , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
The Neurospora crassa leu-1 gene encodes beta-isopropylmalate dehydrogenase (IPMDH; EC 1.1.1.85), an enzyme in the leucine biosynthetic pathway. We determined the nucleotide sequence of the entire leu-1 gene and of four independent cDNA clones. By comparing the genomic and cDNA sequences, four introns were identified in the 5' portion of the gene and a single open reading frame was established. One of the introns is located within the 5'-noncoding region of the transcript. The deduced amino acid sequence encoded by leu-1 was aligned with that of the homologous yeast enzyme and extensive sequence identity was uncovered. The lesion present in a conventional leu-1 mutant was identified as the insertion of a single base pair.
Assuntos
Oxirredutases do Álcool/genética , Neurospora crassa/genética , 3-Isopropilmalato Desidrogenase , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Fúngico , Íntrons , Dados de Sequência Molecular , Neurospora crassa/enzimologia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
The nit-2 gene of Neurospora crassa encodes a trans-acting regulatory protein that activates the expression of a number of structural genes which code for nitrogen catabolic enzymes, including nitrate reductase. The NIT2 protein contains a Cys2/Cys2-type zinc-finger DNA-binding domain that recognizes promoter regions of the Neurospora nitrogen-related genes. The NIT2 zinc-finger domain/beta-Gal fusion protein was shown to recognize and bind in a specific manner to two upstream fragments of the nia gene of Lycopersicon esculentum (tomato) in vitro, whereas two mutant NIT2 proteins failed to bind to the same fragments. The dissociation kinetics of the complexes formed between the NIT2 protein and the Neurospora nit-3 and the tomato nia gene promoters were examined; NIT2 binds more strongly to the nit-3 promoter DNA fragment than it does to fragments derived from the plant nitrate reductase gene itself. The observed specificity of the binding suggests the existence of a NIT2-like homolog which regulates the expression of the nitrate assimilation pathway of higher plants.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Neurospora crassa/genética , Nitrato Redutases/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco , DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Nitrato Redutase , Plantas/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genéticaRESUMO
The repeat induced point mutation (RIP) phenomenon has been used to generate new mutants of nmr, the negative nitrogen regulatory gene in Neurospora crassa. The wild-type nmr gene was cotransformed along with the hygromycin B resistance gene into wild-type cells by selecting for hygromycin B resistance. Following purification of primary transformants using microconidia, many chlorate-sensitive progeny were obtained from crosses to wild-type. Detailed analyses of some of the progeny revealed that we had generated authentic nmr mutants at high frequency. The polymerase chain reaction was used to amplify and clone a fragment of a mutagenized nmr copy from one of the mutants. The nucleotide sequence analysis showed that 14% of the guanine residues have been converted into adenines, resulting in numerous missense and nonsense mutations. The newly created nmr mutants were found suitable for use as host strains in transformation experiments.
Assuntos
Genes Reguladores , Mutação , Neurospora crassa/genética , Nitrogênio , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Dados de Sequência Molecular , Fenótipo , Plasmídeos , Reação em Cadeia da Polimerase , Mapeamento por RestriçãoRESUMO
Uptake of inorganic sulfate in Neurospora crassa is governed by the sulfur regulatory circuit and is under the control of positively and negatively acting regulatory genes. Two genetically and biochemically distinct systems are responsible for the uptake of sulfate from the environment. One of these, sulfate permease II, encoded by the cys-14 gene, functions primarily in mycelia. A defined region of the CYS-14 protein was highly expressed in Escherichia coli and purified. Anti-CYS-14 antibody was produced and used to detect the CYS-14 protein in N. crassa extracts. The CYS-14 protein has an approximate molecular weight of 95K, in agreement with its calculated size based on its predicted amino acid sequence. The steady-state level of the CYS-14 protein is highly regulated in wild-type mycelia and constitutive in an scon-1 mutant, whereas no CYS-14 protein could be detected in a cys-3 mutant. Following the accumulation of the cys-14 mRNA, that reaches its maximum in about 6 h, the CYS-14 protein accumulates to a maximum level in about 8 h after derepression. During conditions of sulfur repression, the CYS-14 protein turns over with a half-life of approximately 2 h. The CYS-14 protein appears to be localized in the plasma membrane, suggesting that it functions as a sulfate ion transporter.
Assuntos
Proteínas Fúngicas , Proteínas de Membrana/genética , Neurospora crassa/metabolismo , Sulfatos/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Western Blotting , Membrana Celular/metabolismo , Escherichia coli/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Genes Reguladores , Proteínas de Membrana/imunologia , Dados de Sequência Molecular , Neurospora crassa/genética , PlasmídeosRESUMO
The complete nucleotide sequence of the cys-14 gene which encodes sulfate permease II, a member of the sulfur regulatory circuit, is presented. The cys-14 gene contains four introns with consensus splice site sequences and is transcribed from four closely spaced initiation sites located approximately 20 bp upstream of the ATG initiation codon. The translated CYS14 protein is composed of 781 amino acids with a molecular weight of 87,037 and contains 12 potential hydrophobic membrane-spanning domains. cys-4 mRNA was found to turn over with a half-life of approximately 15 min, which presumably contributes to the regulation of sulfate permease II function. The cys-14 gene is highly expressed, but only in cells subject to sulfur limitation, and is turned on by the positive-acting CYS3 sulfur regulatory protein. Results are presented which show that CYS3 protein binds with higher affinity to DNA fragments which contain two or three tandem copies of a binding site sequence. Analyses of binding site specificity via mutated binding site elements showed that different regions of the partially symmetrical CYS3 binding site are important for recognition by the CYS3 regulatory protein.
Assuntos
Proteínas de Transporte de Ânions , DNA Fúngico/genética , Proteínas Fúngicas , Genes Fúngicos , Proteínas de Membrana , Proteínas de Membrana Transportadoras/genética , Neurospora crassa/genética , RNA Mensageiro/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Deleção Cromossômica , Clonagem Molecular , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neurospora crassa/enzimologia , Sondas de Oligonucleotídeos , Conformação Proteica , RNA Mensageiro/metabolismo , Mapeamento por Restrição , Transcrição GênicaRESUMO
The genes coding for the branched-chain amino acid biosynthetic enzymes comprise an integrated regulatory system. The expression of the several structural genes coding for enzymes of the isoleucine-valine and leucine pathways is controlled in parallel by the positive-acting regulatory gene, leu-3. The leu-1 and ilv-3 genes, coding for beta-isopropyl-malate dehydrogenase and aceto-hydroxyacid synthase, respectively, were cloned from a cosmid library. Restriction fragment length polymorphism analysis revealed that the two cloned fragments indeed mapped to the genomic locations of the leu-1 and ilv-3 genes, respectively. Northern blot analysis demonstrated that the leu-1 gene is transcribed to give an mRNA of approximately 1.5 kb, whereas the ilv-3 transcript size is 2.6 kb. The expression of both genes appears to be regulated at the transcriptional level. One leu-3 regulatory mutant was greatly deficient in both leu-1 and ilv-3 mRNAs, whereas another leu-3 allele showed an unusual antiparallel pattern of regulation.
Assuntos
Acetolactato Sintase/genética , Oxirredutases do Álcool/genética , Regulação Fúngica da Expressão Gênica , Neurospora crassa/genética , 3-Isopropilmalato Desidrogenase , Clonagem Molecular , Genes Fúngicos , Polimorfismo de Fragmento de Restrição , RNA Fúngico/genética , RNA Mensageiro/genética , Transcrição GênicaRESUMO
The nmr gene is the major negative regulatory gene in the nitrogen control circuit of Neurospora crassa, which, together with positive regulatory genes, governs the expression of multiple unlinked structural genes of the circuit. Possible functional domains of the NMR protein were investigated by mutational analyses using three different approaches. First, the polymerase chain reaction was used to clone the nmr locus from two conventional mutants, V2M304 and MS5, and the mutant amino acid codons were identified. A single point mutation was shown to be responsible for the mutant phenotype in each of these strains. The V2M304 allele contains a nonsense codon, and in the MS5 allele an aspartate has been substituted for glycine at residue 386. Our second approach studied possible functionally important regions in the nmr gene by the use of site-directed mutagenesis. The region containing the naturally occurring substitution in MS5 appears to be essential for function whereas a region in the N-terminal part of the protein does not seem important for NMR function. Finally, over 50% of the protein coding region was randomly mutagenized and amino acid residues that are essential for function and others that are functionally unimportant were identified.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Genes Reguladores , Mutação , Neurospora crassa/genética , Fixação de Nitrogênio/genética , Nitrogênio/metabolismo , Proteínas Repressoras , Alelos , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Códon , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transformação GenéticaRESUMO
In Neurospora the expression of a set of unlinked structural genes, which allows utilization of various nitrogen-containing compounds, is controlled by the positive-acting nit-2 gene and the negative-acting nmr gene. The nucleotide sequence of the nmr gene has been determined and a long open reading frame which encodes a putative protein of 54854 daltons has been identified. A full-length cDNA clone was obtained and its the sequence revealed that the nmr gene contains no introns. The transcriptional start and stop sites have been mapped by S1 nuclease and primer extension. Site-directed mutagenesis was used to introduce stop codons at various locations in the nmr coding region. Transformation assays showed that the proteins lacking up to 16% of the carboxyl-terminus were still functional. Homology searches showed that the nmr protein is homologous to the yeast arginine regulatory gene AR-GRII.
Assuntos
Proteínas Fúngicas/genética , Genes Reguladores , Neurospora crassa/genética , Nitrogênio/metabolismo , Proteínas Repressoras , Sequência de Aminoácidos , Sequência de Bases , Genes Fúngicos , Dados de Sequência Molecular , Mutação , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
There was an overexpression of the c-myc gene (11-fold) and of the c-Ha-ras gene (2-fold) in rat hepatoma 3924A cells compared to normal rat liver as measured by dot-blot analysis of total cytoplasmic RNA. The overexpression of c-myc was attributed to a 10- to 14-fold amplification and rearrangement of the c-myc sequences as determined by Southern blot analysis. The expression of the c-myc also was dependent upon the proliferative state of the hepatoma cells. Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide; NSC 286193), an inhibitor of the activity of IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of GTP biosynthesis, resulted in a rapid drop (less than 1 h) to 50% of control in the target enzyme activity in the hepatoma cells and in a subsequent marked decrease to 55% in GTP concentration. These events were followed at 12 h of tiazofurin treatment by a 3-fold reduction in the expression of the c-myc gene and a 9-fold decline in that of the c-Ha-ras gene. These results in the hepatoma cells provide evidence in support of the earlier demonstrated correlation in K562 cells between GTP concentration and expression of c-myc and c-ras genes (Olah et al., 1989). These genes might depend on GTP for their expression in hepatoma cells and they might cooperate in a signal pathway that controls cell proliferation.
Assuntos
Regulação para Baixo/genética , Expressão Gênica/efeitos dos fármacos , Genes ras/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/genética , Proteínas Proto-Oncogênicas/genética , Ribavirina/farmacologia , Ribonucleosídeos/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Amplificação de Genes/efeitos dos fármacos , Rearranjo Gênico , Genes ras/fisiologia , Guanosina Trifosfato/metabolismo , IMP Desidrogenase/antagonistas & inibidores , IMP Desidrogenase/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Oncogenes/genética , Proteínas Proto-Oncogênicas c-myc , Proteínas Proto-Oncogênicas p21(ras) , Ratos , Ribavirina/análogos & derivadosRESUMO
To investigate the organization of the ribosomal RNA genes in Cephalosporium acremonium, we cloned the whole r X DNA repeat in pBR322 and pNEO plasmids. Both the cloned and the genomic r X DNA fragments were characterized by restriction mapping. The r X DNA repeat unit was found to be 8.0 kb long and there was no significant heterogeneity among the individual repeats.