RESUMEN

Focal adhesion kinase (FAK) is a critical component in transducing signals downstream of both integrins and growth factor receptors. To determine how the loss of FAK affects the epidermis in vivo, we have generated a mouse model with a keratinocyte-restricted deletion of fak (FAKK5 KO mice). FAK(K5 KO) mice displayed three major phenotypes--irregularities of hair cycle, sebaceous glands hypoplasia, and a thinner epidermis--pointing to defects in the proliferative capacity of multipotent stem cells found in the bulge. FAK-null keratinocytes in conventional primary culture undergo massive apoptosis hindering further analyses, whereas the defects observed in vivo do not shorten the mouse lifespan. These results suggest that the structure and the signaling environment of the native tissue may overcome the lack of signaling through FAK. Our findings point to the importance of in vivo and three-dimensional in vitro models in analyses of cell migration, proliferation, and survival. Surprisingly, the difference between FAKloxP/+ and FAKK5 KO mice in wound closure was not statistically significant, suggesting that in vivo loss of FAK does not affect migration/proliferation of basal keratinocytes in the same way as it affects multipotent stem cells of the skin.

Asunto(s)

Quinasa 1 de Adhesión Focal/genética , Cabello/anomalías , Queratinocitos/enzimología , Cicatrización de Heridas , Animales , Movimiento Celular , Proliferación Celular , Células Epidérmicas , Epidermis/anomalías , Epidermis/crecimiento & desarrollo , Femenino , Quinasa 1 de Adhesión Focal/deficiencia , Eliminación de Gen , Cabello/citología , Cabello/crecimiento & desarrollo , Queratina-15 , Queratina-5 , Queratinocitos/citología , Queratinas/metabolismo , Masculino , Ratones , Ratones Noqueados , Glándulas Sebáceas/anomalías , Glándulas Sebáceas/citología , Cicatrización de Heridas/genética

RESUMEN

Virtually all in vitro studies of the effects of rhinovirus on human airway epithelium have used cells grown under conditions known to produce low levels of differentiation. The relevance of the results to native epithelium is questionable. Here we grew primary cultures of human tracheal or nasal epithelium under three conditions. One condition produced pseudostratified, mucociliary cells virtually indistinguishable from native epithelium. The other two conditions produced undifferentiated squamous cells lacking cilia. Cells were infected for 6 h with rhinovirus-16. After a 24-h incubation period, we determined levels of viral RNA in the cells, numbers of infectious viral particles released in the mucosal medium, expression of a variety of epithelial cytokines and other proteins, release of IL-6 and IL-8, and transepithelial electrical resistance and voltage. After infection, levels of viral RNA in the poorly differentiated cells were 30 or 130 times those in the differentiated. Furthermore, expression of mRNA for inflammatory cytokines, release of infectious particles, and release of IL-6 and IL-8 were closely correlated with the degree of viral infection. Thus well-differentiated cells are much more resistant to viral infection and its functional consequences than are poorly differentiated cells from the same source.

Asunto(s)

Resfriado Común/inmunología , Células Epiteliales/virología , Mucosa Nasal/citología , Rhinovirus , Tráquea/citología , Diferenciación Celular , Células Cultivadas , Impedancia Eléctrica , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Inmunidad Innata , Interleucina-6/metabolismo , Interleucina-8/metabolismo , ARN Viral/análisis , Rhinovirus/genética

RESUMEN

Interleukin (IL)-13, a cytokine released by T lymphocytes during immediate hypersensitivity responses, is a central mediator of asthma. Because IL-13 induces phenotypic features of asthma in mice deficient in T and B lymphocytes, it is likely that this cytokine contributes to the development of asthma by acting directly on resident airway cells. To analyze the global effects of IL-13 on gene expression in airway cells that could contribute to the phenotypic features of asthma, we used Genechip HuGene FL arrays (Affymetrix, Santa Clara, CA) that contain probes for approximately 6,500 human genes. Despite activating a common signaling pathway, IL-13 induced dramatically different patterns of gene expression in primary cultures of airway epithelial cells, airway smooth muscle cells, and lung fibroblasts, with little overlap among cell types. The most prominent effects of IL-13 were on airway smooth muscle, but several genes induced in airway epithelial cells and fibroblasts are also candidates that may contribute to phenotypic features of asthma. These results suggest that the in vivo response to IL-13 in the airways likely results from a combination of distinct effects on each of several resident airway cell types.

Asunto(s)

Regulación de la Expresión Génica , Interleucina-13/farmacología , Sistema Respiratorio/citología , Transcripción Genética , Células Cultivadas , Endopeptidasas/efectos de los fármacos , Endopeptidasas/genética , Proteínas de la Matriz Extracelular/efectos de los fármacos , Proteínas de la Matriz Extracelular/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Humanos , Interleucina-13/metabolismo , Canales Iónicos/efectos de los fármacos , Canales Iónicos/genética , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Inhibidores de Proteasas , Mucosa Respiratoria/citología , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/fisiología , Sistema Respiratorio/efectos de los fármacos , Factor de Transcripción STAT6 , Transducción de Señal , Transactivadores/efectos de los fármacos , Transactivadores/genética

RESUMEN

Comprehensive and systematic analysis of airway gene expression represents a strategy for addressing the multiple, complex, and largely untested hypotheses that exist for disease mechanisms, including asthma. Here, we report a novel real-time PCR-based method specifically designed for quantification of multiple low-abundance transcripts using as little as 2.5 fg of total RNA per gene. This method of gene expression profiling has the same specificity and sensitivity as RT-PCR and a throughput level comparable to low-density DNA microarray hybridization. In this two-step method, multiplex RT-PCR is successfully combined with individual gene quantification via real-time PCR on generated cDNA product. Using this method, we measured the expression of 75 genes in bronchial biopsies from asthmatic versus healthy subjects and found expected increases in expression levels of Th2 cytokines and their receptors in asthma. Surprisingly, we also found increased gene expression of NKCC1--a Na+-K+-Cl- cotransporter. Using immunohistochemical method, we confirmed increased protein expression for NKCC1 in the asthmatic subject with restricted localization to goblet cells. These data validate the new transcriptional profiling method and implicate NKCC1 in the pathophysiology of mucus hypersecretion in asthma. Potential applications for this method include transcriptional profiling in limited numbers of laser captured cells and validation of DNA microarray data in clinical specimens.

Asunto(s)

Asma/genética , Bronquios/patología , Proteínas Portadoras/genética , Cloruros/metabolismo , Perfilación de la Expresión Génica/métodos , Potasio/metabolismo , Sodio/metabolismo , Adulto , Resistencia de las Vías Respiratorias/genética , Asma/patología , Bronquios/química , Femenino , Dosificación de Gen , Humanos , Simportadores de Cloruro de Sodio-Potasio , Transcripción Genética/genética

RESUMEN

Excessive airway mucus is an important cause of morbidity and mortality in asthma, but the relationship between accumulation of mucus and goblet cell size, number, and function is incompletely understood. To address these questions, stored mucin in the epithelium and goblet cell size and number were measured morphometrically, and mucin gene expression was measured by polymerase chain reaction and immunohistochemistry in endobronchial biopsies from 13 subjects with mild and moderate asthma and from 12 healthy control subjects. Secreted mucin was measured in induced sputum. We found that stored mucin in the airway epithelium was three times higher than normal in the subjects with asthma (p < 0.005). Goblet cell size was similar in both groups, but goblet cell number was significantly higher in the subjects with asthma (93,043 +/- 15,824 versus 41,959 +/- 9,230/mm3, p < 0.05). In mild asthma (FEV1 > or = 80% pred, n = 7), the level of stored mucin was as high as in moderate asthma (FEV1 < 80% pred, n = 6), but the level of secreted mucin was significantly lower (28.4 +/- 6.3 versus 73.5 +/- 47.5 microg/ml, p < 0.05). Secreted mucin was inversely correlated with stored mucin for the whole asthma group (rs = -0.78, p = 0.007). MUC5AC was the predominant mucin gene expressed in healthy subjects and subjects with asthma, and MUC5AC protein was increased in the subjects with asthma. We conclude that even mild asthma is associated with goblet cell hyperplasia and increased stored mucin in the airway epithelium, whereas moderate asthma is associated with increased stored mucin and secreted mucin. These findings suggest that acute degranulation of hyperplastic goblet cells may represent a mechanism for asthma exacerbations in mild and moderate asthma and that chronic degranulation of goblet cells may contribute to chronic airway narrowing in moderate asthma.

Asunto(s)

Asma/patología , Células Caliciformes/patología , Mucinas/genética , Adulto , Biopsia , Degranulación de la Célula/fisiología , Femenino , Volumen Espiratorio Forzado/fisiología , Expresión Génica/fisiología , Humanos , Hiperplasia , Masculino , Datos de Secuencia Molecular , Mucina 5AC , Mucosa Respiratoria/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

In this report, we describe the identification and molecular characterization of a human RAD50 homolog, hRAD50. hRAD50 was included in a collection of cDNAs which were isolated by a direct cDNA selection strategy focused on the chromosomal interval spanning 5q23 to 5q31. Alterations of the 5q23-q31 interval are frequently observed in myelodysplasia and myeloid leukemia. This strategy was thus undertaken to create a detailed genetic map of that region. Saccharomyces cerevisiae RAD50 (ScRAD50) is one of three yeast RAD52 epistasis group members (ScRAD50, ScMRE11, and ScXRS2) in which mutations eliminate meiotic recombination but confer a hyperrecombinational phenotype in mitotic cells. The yeast Rad50, Mre11, and Xrs2 proteins appear to act in a multiprotein complex, consistent with the observation that the corresponding mutants confer essentially identical phenotypes. In this report, we demonstrate that the human Rad50 and Mre11 proteins are stably associated in a protein complex which may include three other proteins. hRAD50 is expressed in all tissues examined, but mRNA levels are significantly higher in the testis. Other human RAD52 epistasis group homologs exhibit this expression pattern, suggesting the involvement of human RAD52 epistasis group proteins in meiotic recombination. Human RAD52 epistasis group proteins are highly conserved and act in protein complexes that are analogous to those of their yeast counterparts. These findings indicate that the function of the RAD52 epistasis group is conserved in human cells.

Asunto(s)

Cromosomas Humanos Par 5/genética , Reparación del ADN , Endodesoxirribonucleasas , Exodesoxirribonucleasas , Proteínas Fúngicas/metabolismo , Recombinación Genética , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Fibroblastos/metabolismo , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Hígado/metabolismo , Sustancias Macromoleculares , Masculino , Datos de Secuencia Molecular , Complejos Multiproteicos , Ovario/metabolismo , ARN Mensajero/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52 , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Testículo/metabolismo , Timo/metabolismo

RESUMEN

Interleukin-13 (IL-13) and IL-4 are cytokines produced by T cells that are encoded by the q23-31 region of human chromosome 5. To investigate the regulation of IL-13 gene expression by T cells, we isolated and sequenced the human IL-13 gene, analyzed its 5'-flanking region for potential transcriptional activation elements, and examined its expression in nontransformed T-lineage cell populations. The human IL-13 gene was located 12.5-kb upstream of the IL-4 gene and 2-kb downstream of a CpG island. The IL-13 gene 5' flank region included a segment with sequence homology to P elements of the IL-4 promoter involved in transcriptional activation in T cells. Mutation of the IL-13 P element site significantly reduced IL-13 promoter activity in response to T-cell activation. Oligonucleotides containing the IL-13 or IL-4 P element sites specifically bound the transcriptional activator protein, nuclear factor-activated T cells, preformed (NF-ATp), when incubated with nuclear protein extracts from activated T cells. Similar to IL-4, IL-13 mRNA expression was highest in T-cell populations enriched for cells that had previously been primed in vivo or in vitro, indicating that priming increases the expression of the IL-13 and IL-4 genes in a coordinate manner. Because the primed T cells contain higher levels of nuclear NF-ATp, capable of binding to P elements of the IL-4 and IL-13 promoters, than do freshly-isolated T cells, the NF-AT-binding P elements are attractive candidates to mediate the coordinate expression of these two cytokine genes.

Asunto(s)

Cromosomas Humanos Par 5/genética , Regulación de la Expresión Génica , Interleucina-13/genética , Interleucina-4/genética , Proteínas Nucleares , Subgrupos de Linfocitos T/metabolismo , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Ciclosporina/farmacología , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Ligamiento Genético , Humanos , Interleucina-13/biosíntesis , Interleucina-2/biosíntesis , Interleucina-2/genética , Interleucina-4/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Mitógenos/farmacología , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Factores de Transcripción/metabolismo

RESUMEN

The q23-q33 region of human chromosome 5 encodes a large number of growth factors, growth factor receptors, and hormone/neurotransmitter receptors. This is also the general region into which several disease genes have been mapped, including diastrophic dysplasia, Treacher Collins syndrome, hereditary startle disease, the myeloid disorders that are associated with the 5q-syndrome, autosomal-dominant forms of hereditary deafness, and limb girdle muscular dystrophy. We have developed a framework physical map of this region using cosmid clones isolated from the Los Alamos arrayed chromosome 5-specific library. Entry points into this library included 14 probes to genes within this interval and one anonymous polymorphic marker locus. A physical map has been constructed using fluorescence in situ hybridization of these cosmids on metaphase and interphase chromosomes, and this is in good agreement with the radiation hybrid map of the region. The derived order of loci across the region is cen-IL4-IL5-IRF1-IL3-IL9-EGR1-CD1 4-FGFA-GRL-D5S207-ADRB2-SPARC-RPS14+ ++-CSF1R- ADRA1, and the total distance spanned by these loci is approximately 15 Mb. The framework map, genomic clones, and contig expansion within 5q23-q33 should provide valuable resources for the eventual isolation of the clinically relevant loci that reside in this region.

Asunto(s)

Mapeo Cromosómico , Cromosomas Humanos Par 5 , Clonación Molecular , Cósmidos , Humanos , Hibridación Fluorescente in Situ , Masculino , Células Tumorales Cultivadas

RESUMEN

We have developed modifications to direct cDNA selection that allow the rapid and reproducible isolation of low abundance cDNAs encoded by large genomic clones. Biotinylated, cloned genomic DNAs are hybridized in solution with amplifiable cDNAs. The genomic clones and attached cDNAs are captured on streptavidin coated magnetic beads, the cDNAs are eluted and amplified. We have applied this protocol to a 425kb YAC that contains the human IL4 and IL5 genes. After two cycles of enrichment twenty-four cDNAs were evaluated, all of which were homologous to the YAC. DNA sequencing revealed that nine cDNAs were 100% homologous to the interferon regulatory factor 1 (IRF1) gene. Six clones were 70% homologous to the murine P600 gene, which is coexpressed with IL4 and IL5 in mouse Th2 cells. The nine remaining clones were unique within the sequence databases and were non redundant. All of the selected cDNAs were initially present at very low abundance and were enriched by as much as 100,000-fold in two cycles of enrichment. This modified selection technique should be readily applicable to the isolation of many candidate disease loci as well as the derivation of detailed transcription maps across large genomic regions.

Asunto(s)

ADN/genética , ADN/aislamiento & purificación , Genes Reguladores , Interleucina-4/genética , Interleucina-6/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biotina , Southern Blotting , Células Cultivadas , Clonación Molecular , Bases de Datos Factuales , Humanos , Activación de Linfocitos , Linfocitos/inmunología , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Biosíntesis de Proteínas , Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido

RESUMEN

The distal portion of the long arm of human chromosome 5 contains an impressive number of genes encoding growth factors, growth factor receptors, and hormone/neurotransmitter receptors. The order of and relative distance between 18 of these genes was determined by radiation hybrid mapping. There is only a single gap in a contiguous radiation map from 5q22-5q35. For this set of radiation hybrids, one map unit (centiray) corresponds to 20-50 kb of DNA. Close physical proximity for several pairs of loci was predicted by the map. Two sets of these were found to be contained in single YAC clones. The physical map produced by radiation hybrid mapping should prove useful in efforts to identify four disease genes that have been assigned to distal 5q by linkage studies.

Asunto(s)

Cromosomas Humanos Par 5 , Sustancias de Crecimiento/genética , Receptores de Superficie Celular/genética , Secuencia de Bases , Mapeo Cromosómico/métodos , ADN/genética , Marcadores Genéticos , Humanos , Células Híbridas/efectos de la radiación , Datos de Secuencia Molecular , Receptores de Neurotransmisores/genética

RESUMEN

A method is described for the isolation of chromosome region specific cosmids. The 5q35 region of the long arm of human chromosome 5 was microdissected, digested with MboI, ligated to oligonucleotide adaptors, amplified by the polymerase chain reaction and cloned into a plasmid vector. Inserts which did not contain highly repetitive sequences were used to screen a chromosome 5 cosmid library by direct hybridization. There were 33 positive cosmid clones identified with 4 microclones. Individual cosmid clones were biotinylated and used as probes for fluorescence in situ hybridization to metaphase chromosomes. Of the 33 cosmids that were mapped, 29 localized to q35 and 4 to q34, demonstrating the specificity of the microdissection library and the cosmids.

Asunto(s)

Cromosomas Humanos Par 5 , Cósmidos , Secuencia de Bases , Southern Blotting , Clonación Molecular , ADN , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa

RESUMEN

Macrophage colony stimulating factor (CSF-1) is a member of a family of glycoproteins that are necessary for the normal proliferation and differentiation of myeloid progenitor cells. The human CSF-1 gene has previously been assigned to chromosome 5 using somatic cell hybrids, and further localized to 5q33 by in situ hybridization with a 3H labelled cDNA probe. However, the murine macrophage colony stimulating factor gene (csfm) has been localized to a region on mouse chromosome 3 which was previously shown to be syntenic with the proximal region of 1p and not 5q. Using a human genomic DNA clone that contains the CSF-1 gene, we have localized CSF-1 to chromosome 1p13-21 by fluorescence in situ hybridization. The reassignment of the CSF-1 gene argues against its involvement in myeloid disorders with deletions of the long arm of chromosome 5.

Asunto(s)

Artefactos , Cromosomas Humanos Par 1 , Factor Estimulante de Colonias de Macrófagos/genética , Animales , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos Par 5 , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Humanos , Cariotipificación , Metafase , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Espectrometría de Fluorescencia

RESUMEN

Interleukin 3 (encoded by the IL3 gene) and granulocyte-macrophage colony-stimulating factor (encoded by the CSF2 gene) are small secreted polypeptides that bind to specific cell surface receptors and regulate the growth, gene expression, and differentiation of many of the hematopoietic cell lineages, particularly nonlymphoid cells. The IL3 and CSF2 genes have been cloned and mapped to human chromosome bands 5q23-31. Only 10 kilobases of DNA separates the two genes, suggesting that they have a common origin and/or regulation. We have cloned 70 kilobases of genomic DNA that includes the IL3 and CSF2 genes, as well as flanking sequences, and report a physical map of this region. Several unique-sequence DNA segments have been identified in this region, and one of these fragments detects two restriction fragment length polymorphisms in DNA from unrelated Caucasians. Segregation of these DNA polymorphisms was followed in the Centre Etudé du Polymorphisme Humaine (CEPH) panel of 40 large three-generation pedigrees, and linkage was detected with 17 genetic markers previously typed in these families. Multipoint linkage analysis permits the placement of the region containing the IL3 and CSF2 structural genes on the recombination-genetic linkage map of chromosome 5q and thereby allows the role of these genes in leukemogenesis to be more critically examined.

Asunto(s)

Cromosomas Humanos Par 5 , Ligamiento Genético , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Interleucina-3/genética , Polimorfismo de Longitud del Fragmento de Restricción , Secuencia de Bases , Mapeo Cromosómico , Femenino , Biblioteca Genómica , Humanos , Linfocitos/fisiología , Masculino , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Mapeo Restrictivo

RESUMEN

We have identified three new frameshift mutations in the CFTR gene in patients with cystic fibrosis (CF). The first one involves the deletion of an adenine nucleotide in exon 4 in an African-American patient (CF444delA), the second involves the insertion of a cytosine nucleotide in exon 13 in an Italian patient (CF2522insC), and the third results from the deletion of a thymidine nucleotide in exon 19 in a Soviet patient (CF3821delT). Each mutation is predicted to result in premature termination of the CFTR protein.

Asunto(s)

Fibrosis Quística/genética , Mutación del Sistema de Lectura , Proteínas de la Membrana/genética , Adulto , África/etnología , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Preescolar , Fibrosis Quística/etnología , Regulador de Conductancia de Transmembrana de Fibrosis Quística , ADN , Exones , Humanos , Italia/etnología , Datos de Secuencia Molecular , U.R.S.S./etnología , Estados Unidos

Asunto(s)

Mapeo Cromosómico , Factores Estimulantes de Colonias/genética , Sustancias de Crecimiento/genética , Interleucina-3/genética , Southern Blotting , Clonación Molecular , Ligamiento Genético , Genoma Humano , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Humanos

Asunto(s)

Mapeo Cromosómico , Factores Estimulantes de Colonias/genética , Genes , Sustancias de Crecimiento/genética , Interleucina-3/genética , Secuencia de Bases , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Humanos , Datos de Secuencia Molecular , Mapeo Restrictivo

Asunto(s)

Genes , ATPasa Intercambiadora de Sodio-Potasio/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/genética , Enzimas de Restricción del ADN , Humanos , Conformación Proteica , ARN Mensajero/genética

RESUMEN

A procedure for simultaneous large-scale purification of the bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase has been developed. The method involves bacterial cell disruption by sonication, fractionation of cell extract with polymin P, salt elution from the polymin pellets, ammonium sulfate precipitation, and subsequent column chromatography purification of the enzymes. To enrich the enzyme content highly in the initial source non-permissive Escherichia coli B-23 cells infected with T4 amN82 phage were used. The procedure described is rapid, reproducible, high in yield, and able to handle preparations using from 1 g to 200 g cell paste. It can be easily scaled up. The method results in large amounts of the enzymes with very high specific activities, good stability essential lacking exonuclease and endonuclease contamination. The final enzyme preparations were efficiently used in DNA sequencing and in multiple experiments on construction of various recombinant DNAs for cloning and expression in vivo.

Asunto(s)

ADN Ligasas/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Escherichia coli/enzimología , Fosfotransferasas/aislamiento & purificación , Polinucleótido 5'-Hidroxil-Quinasa/aislamiento & purificación , Polinucleótido Ligasas/aislamiento & purificación , ARN Ligasa (ATP)/aislamiento & purificación , Fagos T/enzimología , ADN Ligasas/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Endonucleasas/aislamiento & purificación , Exonucleasas/aislamiento & purificación , Métodos , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , ARN Ligasa (ATP)/metabolismo

RESUMEN

Chemically synthesized leu-enkephalin gene was fused to a large Eco RI-Bam HI fragment of pBR322 along with a Eco RI fragment of Ch4A phage DNA carrying the promoter and most of the E.coli beta-galactosidase gene. The resulting recombinant DNA was used to transform E. coli cells. Transformants were screened for Tc-sensitivity, Am-resistance, and beta-galactosidase constitutional synthesis. Restriction endonuclease analysis combined with DNA sequencing of the plasmid DNAs revealed a complete nucleotide leu-enkephalin sequence and Eco RI lac-operon fragment in two possible orientations. Radioimmunoassay for leu-enkephalin activity in BrCN-treated bacterial extracts showed that in vivo leu-enkephalin is synthesized only in strains carrying plasmids with the proper lac-fragment orientation. About 5.10(4) molecules of the former are synthesized per single E. coli cell. One of the clones was used for leu-enkephalin purification. Using 100 g of cells it is possible to obtain about 2 mg of practically pure leu-enkephalin.Images

Asunto(s)

ADN Recombinante/metabolismo , Endorfinas/aislamiento & purificación , Encefalinas/aislamiento & purificación , Escherichia coli/metabolismo , Cromatografía en Gel , Cromatografía en Papel , Encefalina Leucina , Encefalinas/biosíntesis , Escherichia coli/genética , Plásmidos

Asunto(s)

Clonación Molecular , Endorfinas/genética , Encefalinas/genética , Escherichia coli/genética , Genes , Ingeniería Genética , Secuencia de Bases , ADN Recombinante , Vectores Genéticos , Leucina , Plásmidos