RESUMEN
We have developed Escherichia coli and Pseudomonas expression vectors based on the alkane-responsive Pseudomonas putida (oleovorans) GPo1 promoter PalkB. The expression vectors were tested in several E. coli strains, P. putida GPo12 and P. fluorescens KOB2Delta1 with catechol-2,3-dioxygenase (XylE). Induction factors ranged between 100 and 2700 for pKKPalk in E. coli and pCom8 in Pseudomonas strains, but were clearly lower for pCom8, pCom9, and pCom10 in E. coli. XylE expression levels of more than 10% of total cell protein were obtained for E. coli as well as for Pseudomonas strains.
Asunto(s)
Alcanos/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Escherichia coli/genética , Vectores Genéticos , Oxigenasas de Función Mixta/genética , Regiones Promotoras Genéticas , Pseudomonas fluorescens/genética , Pseudomonas putida/genética , Secuencia de Bases , Citocromo P-450 CYP4A , ADN Bacteriano , Expresión Génica , Datos de Secuencia Molecular , PlásmidosRESUMEN
The Roundabout (Robo) family of receptors and their extracellular ligands, the Slit protein family, play important roles in repulsive axon guidance. First identified in Drosophila, Robo receptors form an evolutionarily conserved sub-family of the immunoglobulin (Ig) superfamily that are characterized by the presence of five Ig repeats and three fibronectin-type III repeats in the extracellular domain, a transmembrane domain, and a cytoplasmic domain with several conserved motifs that play important roles in Robo-mediated signaling (Cell 92 (1998) 205; Cell 101 (2000) 703). Robo family members have now been identified in C. elegans, Xenopus, rat, mouse, and human (Cell 92 (1998) 205; Cell 92 (1998) 217; Cell 96 (1999) 807; Dev. Biol. 207 (1999) 62). Furthermore, multiple robo genes have been described in Drosophila, rat, mouse and humans, raising the possibility of potential redundancy and diversity in robo gene function. As a first step in elucidating the role of Robo receptors during vertebrate development, we identified and characterized two Robo family members from zebrafish. We named these zebrafish genes robo1 and robo3, reflecting their amino acid sequence similarity to other vertebrate robo genes. Both genes are dynamically expressed in the developing nervous system in distinct patterns. robo3 is expressed during the first day of development in the hindbrain and spinal cord and is later expressed in the tectum and retina. robo1 nervous system expression appears later in development and is more restricted. Moreover, both genes are expressed in non-neuronal tissues consistent with additional roles for these genes during development.
Asunto(s)
Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Inmunohistoquímica , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso , Sistema Nervioso/embriología , Filogenia , ARN/metabolismo , Mapeo de Híbrido por Radiación , Homología de Secuencia de Aminoácido , Factores de Tiempo , Distribución Tisular , Proteínas de Pez Cebra , Proteínas RoundaboutRESUMEN
Neurotactin (NRT), a member of the cholinesterase-homologous protein family, is a heterophilic cell adhesion molecule that is required for proper axon guidance during Drosophila development. In this study, we identify amalgam (AMA), a member of the immunoglobulin superfamily, as a ligand for the NRT receptor. Using transfected Schneider 2 cells and embryonic primary cultures, we demonstrate that AMA is a secreted protein. Furthermore, AMA is necessary for NRT-expressing cells both to aggregate with themselves and to associate with embryonic primary culture cells. Aggregation assays performed with truncated NRT molecules reveal that the integrity of the cholinesterase-like extracellular domain was not required either for AMA binding or for adhesion, with only amino acids 347-482 of the extracellular domain being necessary for both activities. Moreover, the NRT cytoplasmic domain is required for NRT-mediated adhesion, although not for AMA binding. Using an ama-deficient stock, we find that ama function is not essential for viability. Pupae deficient for ama do exhibit defasciculation defects of the ocellar nerves similar to those found in nrt mutants.
Asunto(s)
Axones , Adhesión Celular , Proteínas de Drosophila , Drosophila/embriología , Inmunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Inmunoglobulinas/genética , Ligandos , ARN/metabolismoRESUMEN
The Abelson tyrosine kinase (Abl) is integrated into signal transduction networks regulating axon outgrowth. We have identified the Drosophila trio gene through a mutation that exacerbates the Abl mutant phenotype. Drosophila Trio is an ortholog of mammalian Trio, a protein that contains multiple spectrin-like repeats and two Dbl homology (DH) domains that affect actin cytoskeletal dynamics via the small GTPases Rho and Rac. Phenotypic analysis demonstrates that trio and Abl cooperate in regulating axon outgrowth in the embryonic central nervous system (CNS). Dosage-sensitive interactions between trio and Abl, failed axon connections (fax), and enabled (ena) indicate that Trio is integrated into common signaling networks with these gene products. These observations suggest a mechanism by which Abl-mediated signaling networks influence the actin cytoskeleton in neuronal growth cones.
Asunto(s)
Proteínas de Drosophila , Dosificación de Gen , Genes abl/genética , Mutación Missense/fisiología , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Retina/embriología , Animales , Axones/metabolismo , Sistema Nervioso Central/embriología , Sistema Nervioso Central/metabolismo , Drosophila , Genotipo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/fisiología , Datos de Secuencia Molecular , Mutación Missense/genética , Fenotipo , Fosfoproteínas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/fisiología , Retina/metabolismoAsunto(s)
Axones/fisiología , Proteínas de Caenorhabditis elegans , Movimiento Celular/fisiología , Receptores de Superficie Celular/metabolismo , Proteínas Supresoras de Tumor , Animales , Sitios de Unión , Moléculas de Adhesión Celular/metabolismo , Citoplasma/metabolismo , Proteínas del Helminto/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Factores de Crecimiento/metabolismoRESUMEN
We show that the Commissureless (COMM) transmembrane protein is required at neuromuscular synaptogenesis. All muscles in the Drosophila embryo express COMM during the period of motoneuron-muscle interaction. It is endocytosed into muscles before synaptogenesis. In comm loss-of-function mutants, motoneuron growth cones fail to initiate synaptogenesis at target muscles. This stall phenotype is rescued by supplying wild-type COMM to the muscles. Cytoplasmically truncated COMM protein fails to internalize. Expressing this mutant protein in muscles phenocopies the synaptogenesis defects of comm mutants. Thus, synaptogenesis initiation is positively correlated with endocytosis of COMM in postsynaptic muscle cells. We propose that COMM is an essential part of the dynamic cell surface remodeling needed by postsynaptic cells in coordinating synaptogenesis initiation.
Asunto(s)
Proteínas de Drosophila , Drosophila/embriología , Drosophila/genética , Endocitosis/genética , Genes de Insecto , Proteínas de Insectos/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Unión Neuromuscular/embriología , Animales , Drosophila/fisiología , Endocitosis/fisiología , Regulación del Desarrollo de la Expresión Génica , Conos de Crecimiento/fisiología , Inmunohistoquímica , Proteínas de Insectos/fisiología , Proteínas de la Membrana/fisiología , Modelos Neurológicos , Neuronas Motoras/fisiología , Mutación , Proteínas del Tejido Nervioso/fisiología , Unión Neuromuscular/fisiología , Fenotipo , Sinapsis/fisiologíaRESUMEN
Netrins are chemotropic guidance signals that play important roles in circumferential axon guidance in C. elegans and in the developing vertebrate spinal cord. We have identified two Drosophila homologs of this protein family (Netrin-A and Netrin-B). Both Netrins are dynamically expressed throughout embryogenesis, including CNS midline expression at the time of commissure formation. Both Netrin genes map close to each other on the X chromosome, and embryos deficient for this region exhibit defects in commissure formation. This CNS phenotype can be rescued by expression of either Netrin at the CNS midline, confirming an important role for Drosophila Netrins in commissural growth cone guidance. A localized source of Netrin protein at the midline is apparently important for function, since ectopic expression of either Netrin throughout the CNS results in phenotypic defects similar to the loss-of-function phenotype.
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Drosophila/genética , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/genética , Animales , Secuencia de Bases , Sistema Nervioso Central/fisiología , Bandeo Cromosómico , Mapeo Cromosómico , Secuencia Conservada , Proteínas de Drosophila , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes de Insecto/genética , Datos de Secuencia Molecular , Mutación/fisiología , Netrina-1 , Netrinas , Neuritas/fisiología , Homología de Secuencia de Aminoácido , Proteínas Supresoras de Tumor , Cromosoma X/genéticaRESUMEN
The commissureless (comm) gene was identified previously in a large-scale screen for mutations that disrupt CNS axon pathways in Drosophila. The comm gene has a unique mutant phenotype: the complete absence of most axon commissures, while midline cells and other aspects of CNS fate and patterning are left unchanged. Here, we report on the molecular cloning, characterization, and expression of the comm gene. comm encodes a novel protein of 370 amino acids that lacks a signal sequence, has a transmembrane domain, and biochemically copurifies with membranes. COMM mRNA and COMM protein are dynamically expressed during embryogenesis, including by CNS midline glia during the formation of the axon commissures. Anti-COMM antibodies reveal strong staining of organelles likely to include the Golgi complex and endosomes and weaker staining of the cell surface. As commissural growth cones contact and traverse the CNS midline, COMM protein is apparently transferred from midline glia to commissural axons.
Asunto(s)
Axones/fisiología , Sistema Nervioso Central/fisiología , Proteínas de la Membrana/genética , Animales , Secuencia de Bases , Drosophila , Expresión Génica/genética , Inmunohistoquímica , Datos de Secuencia MolecularRESUMEN
Over the past year, several systematic genetic screens designed to identify mutations that specifically disrupt axon pathfinding processes in the Drosophila nervous system have been described. The analysis of mutations isolated in these screens, in concert with ongoing cellular studies and the ever increasing number of identified proteins expressed in the Drosophila nervous system, is providing further insights into the molecular mechanisms of growth cone guidance.
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Axones/fisiología , Drosophila/genética , Drosophila/fisiología , Técnicas Genéticas , Fenómenos Fisiológicos del Sistema Nervioso , Animales , Drosophila/embriología , Neuronas Motoras/fisiología , Mutación , Fibras Nerviosas/fisiología , Proteínas del Tejido Nervioso/fisiología , Vías Nerviosas/fisiología , Unión Neuromuscular/embriologíaRESUMEN
The discovery of the striking positional conservation between the Antennapedia and Bithorax homeotic gene complexes (ANT-C and BX-C) in Drosophila melanogaster and the murine Hox and human HOX clusters has had a substantial impact on our understanding of the evolution of development and its genetic regulation. Structural differences do exist among the mammalian Hox complexes and the ANT-C in D. melanogaster. To gain further insight into the evolutionary changes among these complexes, the ANT-C was cloned in the closely related species, Drosophila pseudoobscura. The overall structure of the ANT-C in D. pseudoobscura is highly similar to its D. melanogaster counterpart; however, two differences in the organization of the ANT-C have been identified. First, the z2 gene, a member of the ANT-C in D. melanogaster, is not present in the D. pseudoobscura ANT-C and is possibly absent from the D. pseudoobscura genome. Second, the orientation of the Deformed gene is inverted in D. pseudoobscura, providing it with a 5' to 3' direction of transcription identical to the remaining ANT-C homeobox genes with the exception of fushi tarazu. These differences demonstrate that subtle changes can occur in ANT-C structure during relatively short periods of evolutionary divergence, although the fundamental organization of the complex is conserved. These observations and others suggest that the complex is not absolutely rigid but that selective pressures have maintained this organization of genes for some functional reason that remains elusive.
Asunto(s)
Drosophila/genética , Genes Homeobox , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Mapeo Cromosómico , Clonación Molecular , Secuencia Conservada , ADN/genética , Drosophila melanogaster/genética , Intrones , Datos de Secuencia Molecular , Familia de Multigenes , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
A new semiselective screen (only female progeny survive) for induced aneuploidy in germ cells of Drosophila melanogaster (referred to as 20/Q56 for the X-chromosome mutation markers in the parental females) has been validated by recovering cold, colchicine and N,N-dimethylnitrosamine (DMN) induced chromosome gain and loss events in females that contain structurally normal chromosomes. In addition, the spontaneous and induced results from the 20/Q56 assay, which identifies gain events at division I and loss events at divisions I and II of meiosis, were compared with a nonselective (all progeny survive) modified mating scheme that identifies gains and losses at both divisions of oogenesis. Females with the same genotypes are treated in the two mating schemes and are then mated with males that contain different marked Y chromosomes. The spontaneous rates of chromosome gains and losses were not significantly different in the two mating schemes (these rates ranged from 0.008 to 0.022%), supporting previous reports that spontaneous aneuploidy occurs at a higher frequency at division I of meiosis in females of D. melanogaster than at division II. Both the 20/Q56 and modified screens were able to identify significant increases in aneuploidy after adult treatments with cold shock (10 degrees C and 5 degrees C), colchicine (5 ppm and 10 ppm), and DMN (100 ppm). Brood analysis (five 2-day or five 3-day broods) showed that the largest increases in aneuploidy after cold treatment occurred in the first brood, which contains a high proportion of stage 14 oocytes, whereas colchicine induced the highest frequencies in the latter broods and DMN was effective in all but the last brood. Although the 20/Q56 mating scheme identifies gain events only in division I of meiosis whereas the modified mating scheme identifies gains in both divisions, the 20/Q56 scheme is just as effective in identifying induced aneuploidy as is the modified scheme. There were no significant differences in the frequencies of induced gains or losses in the two schemes. These results also suggest that the 3 treatments induced chromosome gain events mainly at division I of oogenesis. Taken together, the results from this study suggest that the 20/Q56 mating scheme in D. melanogaster, which is semiselective and therefore less expensive and time-consuming to perform, is an appropriate test system to screen for chemical induced aneuploidy in germ cells of a higher organism.
Asunto(s)
Aneuploidia , Drosophila melanogaster/genética , Pruebas Genéticas/métodos , Oocitos/citología , Animales , Cromosomas/efectos de los fármacos , Colchicina/farmacología , Cruzamientos Genéticos , Dimetilnitrosamina/farmacología , Frecuencia de los Genes , Oocitos/efectos de los fármacos , Oogénesis , TemperaturaRESUMEN
The specification of anterior positional information during Drosophila embryogenesis is largely dependent upon the function of the maternal-effect gene bicoid (bcd). Two aspects of bcd function are particularly striking. First, the bcd protein product forms a gradient during early embryogenesis, which regulates the transcription of at least one zygotic segmentation gene, hunchback, in a concentration dependent manner. Secondly, formation of the bcd protein gradient is dependent upon the specific localization of bcd mRNAs at the anterior end of the oocyte/embryo during oogenesis, a process which requires a cis-acting 625 nucleotide sequence within the 3' untranslated region of the bcd mRNA. We have cloned and sequenced the bcd gene from Drosophila pseudoobscura as a tool in identifying important functional domains within this transcription unit. DNA sequence comparisons reveal: (i) varying degrees of amino acid sequence conservation among the proposed functional domains of the bcd protein, (ii) the conservation of potential RNA secondary structures within the bcd mRNA localization element, and (iii) the maintenance of a short open reading frame within the 5' untranslated leader that may play a role in translational regulation. Finally, the D.pseudoobscura bcd gene partially rescues the phenotype of a bcd- mutation when placed into the D.melanogaster genome by germline transformation. The lack of full phenotypic rescue can be explained in part by the observed improper localization of the D.pseudoobscura bcd mRNA when expressed in D.melanogaster.
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Drosophila/genética , Genes Homeobox , ARN Mensajero/genética , Secuencia de Aminoácidos , Animales , Composición de Base , Secuencia de Bases , Clonación Molecular , Drosophila/embriología , Drosophila melanogaster/genética , Biblioteca de Genes , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Fenotipo , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
The immunoglobulin superfamily is a diverse group of proteins that are involved in various aspects of cell surface recognition. Here, we report the characterization of amalgam (ama), a gene in the Antennapedia complex (ANT-C) of D. melanogaster that exhibits amino acid similarity to vertebrate neural cell adhesion molecules and other members of the immunoglobulin superfamily. The putative 333 amino acid ama protein consists of a signal sequence, three immunoglobulin-like domains, and a short slightly hydrophobic carboxy-terminal region. Antibodies against the ama protein reveal that it accumulates on the surface of various mesodermal and neural cells during embryogenesis. The function of this protein remains elusive, as no mutations have been recovered for ama during saturation EMS mutagenesis of this chromosomal region.