RESUMEN

Genome-wide chemical mutagenesis screens in the zebrafish (Danio rerio) have led to the identification of novel genes affecting vertebrate erythropoiesis. In determining if this approach could also be used to clarify the molecular genetics of myelopoiesis, it was found that the developmental hierarchy of myeloid precursors in the zebrafish kidney is similar to that in human bone marrow. Zebrafish neutrophils resembled human neutrophils, possessing segmented nuclei and myeloperoxidase-positive cytoplasmic granules. The zebrafish homologue of the human myeloperoxidase (MPO) gene, which is specific to cells of the neutrophil lineage, was cloned and used to synthesize antisense RNA probes for in situ hybridization analyses of zebrafish embryos. Granulocytic cells expressing zebrafish mpo were first evident at 18 hours after fertilization (hpf) in the posterior intermediate cell mass (ICM) and on the anterior yolk sac by 20 hpf. By 24 hpf, mpo-expressing cells were observed along the ICM and within the developing vascular system. Thus, the mpo gene should provide a useful molecular probe for identifying zebrafish mutants with defects in granulopoiesis. The expression of zebrafish homologues was also examined in 2 other mammalian hematopoietic genes, Pu.1, which appears to initiate a commitment step in normal mammalian myeloid development, and L-Plastin, a gene expressed by human monocytes and macrophages. The results demonstrate a high level of conservation of the spatio-temporal expression patterns of these genes between zebrafish and mammals. The morphologic and molecular genetic evidence presented here supports the zebrafish as an informative model system for the study of normal and aberrant human myelopoiesis. (Blood. 2001;98:643-651)

Asunto(s)

Leucopoyesis/genética , Peroxidasa/genética , Pez Cebra/fisiología , Secuencia de Aminoácidos , Animales , Embrión no Mamífero/enzimología , Embrión no Mamífero/metabolismo , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Granulocitos/enzimología , Granulocitos/fisiología , Humanos , Hibridación in Situ , Glicoproteínas de Membrana , Proteínas de Microfilamentos , Modelos Animales , Datos de Secuencia Molecular , Neutrófilos/enzimología , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas/genética , Sondas ARN/síntesis química , Alineación de Secuencia , Homología de Secuencia , Transactivadores/genética

Asunto(s)

Antiportadores/genética , Eritropoyesis/genética , Mutación , Pez Cebra/genética , Animales , Antiportadores de Cloruro-Bicarbonato , Clonación Molecular , Modelos Animales , Modelos Genéticos

RESUMEN

Spectrins are key cytoskeleton proteins with roles in membrane integrity, cell morphology, organelle transport and cell polarity of varied cell types during development. Defects in erythroid spectrins in humans result in congenital hemolytic anemias with altered red cell morphology. Although well characterized in mammals and invertebrates, analysis of the structure and function of non-mammalian vertebrate spectrins has been lacking. The zebrafish riesling (ris) suffers from profound anemia, where the developing red cells fail to assume terminally differentiated erythroid morphology. Using comparative genomics, erythroid beta-spectrin (sptb) was identified as the gene mutated in ris. Zebrafish Sptb shares 62.3% overall identity with the human ortholog and phylogenetic comparisons suggest intragenic duplication and divergence during evolution. Unlike the human and murine orthologs, the pleckstrin homology domain of zebrafish Sptb is not removed in red cells by alternative splicing. In addition, apoptosis and abnormal microtubule marginal band aggregation contribute to hemolysis of mutant erythrocytes, which are features not present in mammalian red cells with sptb defects. This study presents the first genetic characterization of a non-mammalian vertebrate sptb and demonstrates novel features of red cell hemolysis in non-mammalian red cells. Further, we propose that the distinct mammalian erythroid morphology may have evolved from specific modifications of Sptb structure and function.

Asunto(s)

Eritrocitos/metabolismo , Evolución Molecular , Enfermedades de los Peces/genética , Espectrina/genética , Esferocitosis Hereditaria/veterinaria , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Apoptosis , Secuencia de Bases , ADN Complementario , Eritrocitos/citología , Hemólisis , Datos de Secuencia Molecular , Morfogénesis , Filogenia , Espectrina/fisiología , Esferocitosis Hereditaria/genética

RESUMEN

The zebrafish (Danio rerio) has emerged in recent years as an exciting animal model system for studying vertebrate organ development and, in particular, the development of the hematopoietic system. The combined advantages of developmental biology and genetic screens for mutations in zebrafish have provided insights into early events in hematopoiesis and identified several genes required for normal blood development in vertebrates. As a result of the large-scale mutagenesis screens for developmental mutants, several zebrafish mutants with defects in blood development have been recovered. This review discusses how these blood mutations in zebrafish have given new perspectives on hematopoietic development.

Asunto(s)

Pez Cebra/sangre , Pez Cebra/genética , Animales , Modelos Animales de Enfermedad , Hematopoyesis/genética , Hematopoyesis/fisiología , Mutación

RESUMEN

Defects in iron absorption and utilization lead to iron deficiency and overload disorders. Adult mammals absorb iron through the duodenum, whereas embryos obtain iron through placental transport. Iron uptake from the intestinal lumen through the apical surface of polarized duodenal enterocytes is mediated by the divalent metal transporter, DMTi. A second transporter has been postulated to export iron across the basolateral surface to the circulation. Here we have used positional cloning to identify the gene responsible for the hypochromic anaemia of the zebrafish mutant weissherbst. The gene, ferroportin1, encodes a multiple-transmembrane domain protein, expressed in the yolk sac, that is a candidate for the elusive iron exporter. Zebrafish ferroportin1 is required for the transport of iron from maternally derived yolk stores to the circulation and functions as an iron exporter when expressed in Xenopus oocytes. Human Ferroportin1 is found at the basal surface of placental syncytiotrophoblasts, suggesting that it also transports iron from mother to embryo. Mammalian Ferroportin1 is expressed at the basolateral surface of duodenal enterocytes and could export cellular iron into the circulation. We propose that Ferroportin1 function may be perturbed in mammalian disorders of iron deficiency or overload.

Asunto(s)

Proteínas Portadoras/genética , Proteínas de Transporte de Catión , Evolución Molecular , Hierro/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Paseo de Cromosoma , Clonación Molecular , Embrión no Mamífero/metabolismo , Enterocitos/metabolismo , Eritrocitos/metabolismo , Hemoglobinas/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Hierro/sangre , Ratones , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Fenotipo , Placenta/metabolismo , Homología de Secuencia de Aminoácido , Distribución Tisular , Xenopus , Saco Vitelino/metabolismo , Pez Cebra

RESUMEN

Members of the JAK family of protein tyrosine kinase (PTK) proteins are required for the transmission of signals from a variety of cell surface receptors, particularly those of the cytokine receptor family. JAK function has been implicated in hematopoiesis and regulation of the immune system, and recent data suggest that the vertebrate JAK2 gene may play a role in leukemia. We have isolated and characterized jak cDNAs from the zebrafish Danio rerio. The zebrafish genome possesses 2 jak2 genes that occupy paralogous chromosome segments in the zebrafish genome, and these segments conserve syntenic relationships with orthologous genes in mammalian genomes, suggesting an ancient duplication in the zebrafish lineage. The jak2a gene is expressed at high levels in erythroid precursors of primitive and definitive waves and at a lower level in early central nervous system and developing fin buds. jak2b is expressed in the developing lens and nephritic ducts, but not in hematopoietic tissue. The expression of jak2a was examined in hematopoietic mutants and found to be disrupted in cloche and spadetail, suggesting an early role in hematopoiesis. Taken together with recent gene knockout data in the mouse, we suggest that jak2a may be functionally equivalent to mammalian Jak2, with a role in early erythropoiesis.

Asunto(s)

Eritropoyesis , Regulación del Desarrollo de la Expresión Génica , Genes , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas , Pez Cebra/genética , Alelos , Secuencia de Aminoácidos , Animales , Clonación Molecular , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Inducción Enzimática , Células Precursoras Eritroides/enzimología , Eritropoyesis/genética , Evolución Molecular , Hematopoyesis/genética , Células Madre Hematopoyéticas/enzimología , Humanos , Janus Quinasa 2 , Mamíferos/genética , Mamíferos/metabolismo , Ratones , Datos de Secuencia Molecular , Mutación , Fenotipo , Proteínas Tirosina Quinasas/fisiología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Transcripción Genética , Pez Cebra/embriología

RESUMEN

Transcription factors of the STAT family are required for cellular responses to multiple signaling molecules. After ligand binding-induced activation of cognate receptors, STAT proteins are phosphorylated, hetero- or homodimerize, and translocate to the nucleus. Subsequent STAT binding to specific DNA elements in the promoters of signal-responsive genes alters the transcriptional activity of these loci. STAT function has been implicated in the transduction of signals for growth, reproduction, viral defense, and immune regulation. We have isolated and characterized two STAT homologs from the zebrafish Danio rerio. The stat3 gene is expressed in a tissue-restricted manner during embryogenesis, and larval development with highest levels of transcript are detected in the anterior hypoblast, eyes, cranial sensory ganglia, gut, pharyngeal arches, cranial motor nuclei, and lateral line system. In contrast, the stat1 gene is not expressed during early development. The stat3 gene maps to a chromosomal position syntenic with the mouse and human STAT3 homologs, whereas the stat1 gene does not. Despite a higher rate of evolutionary change in stat1 relative to stat3, the stat1 protein rescues interferon-signaling functions in a STAT1-deficient human cell line, indicating that cytokine-signaling mechanisms are likely to be conserved between fish and tetrapods. Dev Dyn 1999;215:352-370.

Asunto(s)

Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Sistema Nervioso Central/metabolismo , Mapeo Cromosómico , Cromosomas , Secuencia Conservada , Proteínas de Unión al ADN/análisis , Embrión no Mamífero/anatomía & histología , Evolución Molecular , Humanos , Janus Quinasa 1 , Modelos Genéticos , Datos de Secuencia Molecular , Familia de Multigenes , Sistema Nervioso Periférico/metabolismo , Filogenia , Polimorfismo Genético , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución Tisular , Transactivadores/análisis , Transfección , Proteínas de Pez Cebra

Asunto(s)

Técnicas de Cultivo de Célula/métodos , Fibroblastos/citología , Animales , Células Cultivadas , Pez Cebra

RESUMEN

Many human anaemias are caused by defects in haemoglobin synthesis. The zebrafish mutant sauternes (sau) has a microcytic, hypochromic anaemia, suggesting that haemoglobin production is perturbed. During embryogenesis, sau mutants have delayed erythroid maturation and abnormal globin gene expression. Using positional cloning techniques, we show that sau encodes the erythroid-specific isoform of delta-aminolevulinate synthase (ALAS2; also known as ALAS-E), the enzyme required for the first step in haem biosynthesis. As mutations in ALAS2 cause congenital sideroblastic anaemia (CSA) in humans, sau represents the first animal model of this disease.

Asunto(s)

5-Aminolevulinato Sintetasa/genética , Anemia Sideroblástica/enzimología , Anemia Sideroblástica/genética , Isoenzimas/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Anemia Sideroblástica/congénito , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Modelos Animales de Enfermedad , Hemoglobinas/biosíntesis , Hemoglobinas/genética , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Fenotipo , Homología de Secuencia de Aminoácido

RESUMEN

Radiation hybrid panels are already available for genome mapping in human and mouse. In this study we have used two model organisms (chicken and zebrafish) to show that hybrid panels that contain a full complement of the donor genome can be generated by fusion to hamster cells. The quality of the resulting hybrids has been assessed using PCR and FISH. We confirmed the utility of our panels by establishing the percentage of donor DNA present in the hybrids. Our hybrid resources will allow inexpensive gene mapping and we expect that this technology can be transferred to many other species. Such successes are providing the basis for a new era of mapping tools, in the form of whole genome radiation hybrid panels, and are opening new possibilities for systematic genome analysis in the animal genetics community.

Asunto(s)

Células Híbridas/efectos de la radiación , Animales , Línea Celular , Pollos , Mapeo Cromosómico , Cricetinae , Hibridación Fluorescente in Situ , Reacción en Cadena de la Polimerasa , Pez Cebra

RESUMEN

The modification of yeast artificial chromosomes through homologous recombination has become a useful genetic tool for studying gene function and enhancer/promoter activity. However, it is difficult to purify intact yeast artificial chromosome DNA at a concentration sufficient for many applications. Bacterial artificial chromosomes (BACs) are vectors that can accommodate large DNA fragments and can easily be purified as plasmid DNA. We report herein a simple procedure for modifying BACs through homologous recombination using a targeting construct containing properly situated Chi sites. To demonstrate a usage for this technique, we modified BAC clones containing the zebrafish GATA-2 genomic locus by replacing the first coding exon with the green fluorescent protein (GFP) reporter gene. Molecular analyses confirmed that the modification occurred without additional deletions or rearrangements of the BACs. Microinjection demonstrated that GATA-2 expression patterns can be recapitulated in living zebrafish embryos by using these GFP-modified GATA-2 BACs. Embryos microinjected with the modified BAC clones were less mosaic and had improved GFP expression in hematopoietic progenitor cells compared with smaller plasmid constructs. The precise modification of BACs through Chi-stimulated homologous recombination should be useful for studying gene function and regulation in cultured cells or organisms where gene transfer is applicable.

Asunto(s)

Animales Modificados Genéticamente , Proteínas de Unión al ADN/fisiología , Vectores Genéticos , Factores de Transcripción/fisiología , Animales , Cromosomas Bacterianos , Factor de Transcripción GATA2 , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Microinyecciones , Recombinación Genética

RESUMEN

SCL/Tal-1 is a transcription factor necessary for hematopoietic stem cell differentiation. Although SCL is also expressed in endothelial and neural progenitors, SCL function in these cells remains unknown. In the zebrafish mutant cloche (clo), SCL expression is nearly abolished in hematopoietic and vascular tissues. Correspondingly, it was shown previously that clo fails to differentiate blood and angioblasts. Genetic analysis demonstrates that the clo mutation is not linked to the SCL locus. Forced expression of SCL in clo embryos rescues the blood and vascular defects, suggesting that SCL acts downstream of clo to specify hematopoietic and vascular differentiation.

Asunto(s)

Proteínas de Unión al ADN/genética , Endotelio Vascular/embriología , Células Madre Hematopoyéticas/fisiología , Proteínas Proto-Oncogénicas , Factores de Transcripción , Proteínas de Pez Cebra , Pez Cebra/embriología , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Mapeo Cromosómico , Secuencia Conservada , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia Molecular , Mutación , Proteína 1 de la Leucemia Linfocítica T Aguda

RESUMEN

Two CpG mutations at codon 504 of the gene encoding the alpha-subunit of beta-hexosaminidase (the HEX A gene) have been identified previously: (1) a C deletion resulting in premature termination of the alpha-subunit and (2) a G----A transition resulting in 504Arg----His substitution, in patients with infantile Tay-Sachs disease and juvenile GM2 gangliosidosis, respectively. This prompted a search for a C----T transition in the same dinucleotide, as would be expected from the mechanism of CpG mutagenesis. Such a mutation, which results in a substitution of cysteine for arginine, was found in a patient with chronic GM2 gangliosidosis, in compound heterozygosity with the known 269Gly----Ser allele. The biochemical phenotype of the 504Arg----Cys mutation was examined by site-directed mutagenesis of the alpha-subunit cDNA and transfection of Cos-1 cells. The expression of the mutagenized cDNA with the cysteine substitution gave rise to an alpha-subunit with the same defects as those resulting from expression of mutagenized cDNA with the histidine substitution--i.e., secretion primarily as the alpha-monomer rather than as the alpha alpha dimer, along with absence of enzymatic activity. The 504Arg----Cys/269Gly----Ser genotype of the chronic GM2 gangliosidosis patient is shared by her sibling, who has mild adult-onset GM2 gangliosidosis, implying that the clinical differences between them must be attributed to other factors. The family is unique in yet another respect--namely, that the normal allele of the mother and of a 504Arg----Cys heterozygous sibling has a silent mutation, a G----A transition in the wobble position of the glutamic acid codon at position 506.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Codón , Fosfatos de Dinucleósidos/genética , Mutación , beta-N-Acetilhexosaminidasas/genética , Alelos , Secuencia de Bases , Células Cultivadas , Genotipo , Hexosaminidasa A , Humanos , Datos de Secuencia Molecular , Fenotipo , Plásmidos , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Transfección

RESUMEN

Mutations in the HEX A gene, encoding the alpha-subunit of beta-hexosaminidase A (Hex A), are the cause of Tay-Sachs disease as well as of juvenile, chronic, and adult GM2 gangliosidoses. We have examined the distribution of three mutations--a 4-nucleotide insertion in exon 11, a G----C transversion at a 5' splice site in intron 12, and a 269Gly----Ser amino acid substitution in exon 7--among individuals enzymatically diagnosed as carriers of Hex A deficiency. Mutation analysis included polymerase chain reaction (PCR) amplification of the relevant regions of genomic DNA, followed by allele-specific oligonucleotide hybridization; another test for heterozygosity of the exon 11 insertion was based on the formation of heteroduplex PCR fragments of low electrophoretic mobility. The percentage distribution of the exon 11, intron 12, exon 7, and unidentified mutant alleles was 73:15:4:8 among 156 Jewish carriers of Hex A deficiency and 16:0:3:81 among 51 non-Jewish carriers. Regardless of the mutation, the ancestral origin of the Jewish carriers was primarily eastern and (somewhat less often) central Europe, whereas for the non-Jewish carriers it was western Europe. Because a twelfth of the Jewish carriers and four-fifths of the non-Jewish carriers of Hex A deficiency had mutant alleles other than the three common ones tested, enzyme-based tests cannot be replaced by DNA-based tests at the present time. However, DNA-based tests for two-carrier couples could identify those at risk for the chronic/adult GM2 gangliosidoses rather than for infantile Tay-Sachs disease.

Asunto(s)

Heterocigoto , Mutación , Enfermedad de Tay-Sachs/genética , Alelos , Secuencia de Bases , ADN/genética , Análisis Mutacional de ADN , Electroforesis en Gel de Poliacrilamida , Exones , Frecuencia de los Genes , Tamización de Portadores Genéticos , Pruebas Genéticas , Humanos , Intrones , Judíos/genética , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa

RESUMEN

Juvenile GM2 gangliosidosis is a rare neurodegenerative disorder closely related to Tay-Sachs disease but of later onset and more protracted course. The biochemical defect lies in the alpha-subunit of the lysosomal enzyme beta-hexosaminidase. Cultured fibroblasts derived from patient A synthesized an alpha-subunit which could acquire mannose 6-phosphate and be secreted, but which failed to associate with the beta-subunit to form the enzymatically active heterodimer. By contrast, fibroblasts from patient B synthesized an alpha-subunit that was retained in the endoplasmic reticulum. To identify the molecular basis of the disorder, RNA from fibroblasts of these two patients was reverse-transcribed, and the cDNA encoding the alpha-subunit of beta-hexosaminidase was amplified by the polymerase chain reaction (PCR) in four overlapping fragments. The PCR fragments were subcloned and shown by sequence analysis to contain a G to A transition corresponding to substitution of histidine for arginine at position 504 in the case of patient A and at position 499 in the case of patient B. The mutations were confirmed by hybridization of allele-specific oligonucleotides to PCR-amplified fragments of DNA corresponding to exon 13 of the alpha-subunit gene. The Arg504----His mutation was found on both alleles of patient A as well as of another unrelated patient; the homozygosity of this mutant allele is attributable to consanguinity in the two families. The Arg499----His mutation was found in patient B in compound heterozygosity with a common infantile Tay-Sachs allele. There is additional heterogeneity in juvenile GM2 gangliosidosis, as neither mutation was found in the DNA of a fourth patient. The Arg----His mutations at positions 499 and 504 are located at CpG dinucleotides, which are known to be mutagenic "hot spots."

Asunto(s)

Arginina , Gangliosidosis/enzimología , Histidina , Lisosomas/enzimología , Mutación , beta-N-Acetilhexosaminidasas/genética , Secuencia de Bases , Células Cultivadas , Niño , Preescolar , ADN/genética , Femenino , Fibroblastos/enzimología , Gangliósido G(M2) , Gangliosidosis/genética , Homocigoto , Humanos , Sustancias Macromoleculares , Masculino , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Transcripción Genética

RESUMEN

Chronic and adult-onset GM2 gangliosidoses are neurological disorders caused by marked deficiency of the A isoenzyme of beta-hexosaminidase; they occur in the Ashkenazi Jewish population, though less frequently than classic (infantile) Tay-Sachs disease. Earlier biosynthetic studies had identified a defective alpha-subunit that failed to associate with the beta-subunit. We have now found a guanosine to adenosine transition at the 3' end of exon 7, which causes substitution of serine for glycine at position 269 of the alpha-subunit [designated 269 (Gly----Ser) substitution]. An RNase protection assay was used to localize the mutation to a segment of mRNA from fibroblasts of a patient with the adult-onset disorder. That segment of mRNA (after reverse transcription) and a corresponding segment of genomic DNA were amplified by the polymerase chain reaction and sequenced by the dideoxy method. The sequence analysis, together with an assay based on the loss of a ScrFI restriction site, showed that the patient was a compound heterozygote who had inherited the 269 (Gly----Ser) mutation from his father and an allelic null mutation from his mother. The 269 (Gly----Ser) mutation, in compound heterozygosity with a presumed null allele, was also found in fetal fibroblasts with an association-defective phenotype and in cells from five patients with chronic GM2 gangliosidosis. It was not found in beta-hexosaminidase A-deficient cells obtained from patients with infantile Tay-Sachs disease nor in cells from individuals who do not have beta-hexosaminidase A deficiency. However, there must be additional mutations with similar consequences, since the 269 (Gly----Ser) substitution was not present in fibroblasts from two patients with juvenile GM2 gangliosidosis even though these had an association-defective alpha-subunit.

Asunto(s)

Gangliosidosis/genética , Glicina , Judíos/genética , Mutación , Serina , beta-N-Acetilhexosaminidasas/genética , Adulto , Secuencia de Bases , Células Cultivadas , Europa (Continente)/etnología , Fibroblastos/enzimología , Gangliósido G(M2)/genética , Gangliosidosis/enzimología , Humanos , Isoenzimas/genética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Valores de Referencia

RESUMEN

Tay-Sachs disease is a biochemically heterogeneous lysosomal storage disorder caused by lack of the A isoenzyme of beta-hexosaminidase; the underlying defect is a mutation in the gene encoding the alpha-chain. It has been shown that fibroblasts isolated from Tay-Sachs patients of Ashkenazi Jewish origin contain no alpha-chain mRNA detectable on Northern blots. We now have compared run-on transcription in nuclei isolated from three strains of Ashkenazi Tay-Sachs fibroblasts and from a strain of normal (IMR90) cells. Using alpha-chain and beta-chain cDNAs as probes, we found no difference in the relative amount of [32P]ribonucleotide added to nascent transcripts; the average ratio of alpha/beta hybridizable radioactivity was 1.3 and 1.4 for mutant and normal cells, respectively. The identity of the Tay-Sachs alpha-chain transcript was confirmed by competition hybridization with excess alpha-chain mRNA. The results indicate that the Ashkenazi Tay-Sachs mutation permits a normal level of transcription of the alpha-chain gene and points to a posttranscriptional defect, such as RNA processing, transport, or stability.

Asunto(s)

Enfermedad de Tay-Sachs/genética , Transcripción Genética , beta-N-Acetilhexosaminidasas/genética , Fibroblastos/enzimología , Humanos , Mutación , Hibridación de Ácido Nucleico , Poli A/análisis , ARN/análisis , ARN Mensajero , Enfermedad de Tay-Sachs/enzimología

RESUMEN

The enzyme N epsilon-hydroxylysine acetylase has been isolated from Escherichia coli 294 carrying recombinant plasmid ABN11. Activity of the enzyme was followed by measurement of the rate of appearance of 2-nitro-5-thiobenzoate, the product of cleavage of 5,5'-dithiobis(2-nitrobenzoate) by free coenzyme A released from its acetyl derivative. The enzyme bound firmly to Reactive Blue 2-Sepharose CL-6B and was eluated with 1.5 M KCl. The protein gave a single band, corresponding to a Mr of 33,000, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In contrast, gel filtration of the native enzyme gave a Mr of 150,000-200,000. A sequence analysis of the DNA at the junction of the first and second genes in the aerobactin operon, considered in conjunction with the N-terminal amino acid sequence of the isolated protein, enabled the conclusion that the acetylase is specified by the second gene in the complex. The enzyme transfers the acetyl moiety from acetyl coenzyme A to a variety of hydroxylamines, with N epsilon-hydroxylysine as the preferred substrate. In agreement with the results found by affinity chromatography, Coomassie Blue was observed to act as a potent inhibitor.

Asunto(s)

Acetiltransferasas/aislamiento & purificación , Escherichia coli/enzimología , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Cinética , Peso Molecular , Colorantes de Rosanilina/farmacología , Especificidad por Sustrato

RESUMEN

The iron-regulated aerobactin operon, about 8 kilobase pairs in size, of the Escherichia coli plasmid ColV-K30 was shown by deletion and subcloning analyses to consist of at least five genes for synthesis (iuc, iron uptake chelate) and transport (iut, iron uptake transport) of the siderophore. The gene order iucABCD iutA was established. The genes were mapped within restriction nuclease fragments of a cloned 16.3-kilobase-pair HindIII fragment. Stepwise deletion and subsequent minicell analysis of the resulting plasmids allowed assignment of four of the five genes to polypeptides of molecular masses 63,000, 33,000 53,000, and 74,000 daltons, respectively. The 74-kilodalton protein, the product of gene iutA, is the outer membrane receptor for ferric aerobactin, whereas the remaining three proteins are involved in biosynthesis of aerobactin. The 33-kilodalton protein, the product of gene iucB, was identified as N epsilon-hydroxylysine:acetyl coenzyme A N epsilon-transacetylase (acetylase) by comparison of enzyme activity in extracts from various deletion mutants. The 53-kilodalton protein, the product of gene iucD, is required for oxygenation of lysine. The 63-kilodalton protein, the product of gene iucA, is assigned to the first step of the aerobactin synthetase reaction. The product of gene iucC, so far unidentified, performs the second and final step in this reaction. This is based on the chemical characterization of two precursor hydroxamic acids (N epsilon-acetyl-N epsilon-hydroxylysine and N alpha-citryl-N epsilon-acetyl-N epsilon-hydroxylysine) isolated from a strain carrying a 0.3-kilobase-pair deletion in the iucC gene. The results support the existence of a biosynthetic pathway in which aerobactin arises by oxygenation of lysine, acetylation of the N epsilon-hydroxy function, and condensation of 2 mol of the resulting aminohydroxamic acid with citric acid.

Asunto(s)

Plásmidos de Bacteriocinas , Escherichia coli/genética , Genes Bacterianos , Ácidos Hidroxámicos/metabolismo , Quelantes del Hierro/metabolismo , Plásmidos , Acetiltransferasas/genética , Proteínas Bacterianas/genética , Transporte Biológico , Genes , Oxigenasas de Función Mixta/genética , Peso Molecular , Operón , Oxo-Ácido-Liasas/genética , Sideróforos