RESUMEN
Inducible, vector-based, expression systems that allow fine control of transgene expression are gaining more and more use in fundamental research as well as in therapeutic applications. In an effort to develop a tightly regulated heterologous expression system for Drosophila Schneider 2 cells, three different inducible reporter constructs were compared. These comprised six copies of the glucocorticoid response element fused to one of three distinct types of Drosophila gene promoters: (1) a TATA-box containing, (2) a TATA-less and (3) a bidirectional core sequence. These were fused to a luciferase reporter gene. The promoter constructs displayed different basal as well as agonist-induced activities. The implications of the observations made are discussed in the context of promoter properties and of induction of genes that may be studied in Drosophila.
Asunto(s)
Drosophila melanogaster/genética , Regulación de la Expresión Génica/genética , Genes Reporteros/genética , Vectores Genéticos/genética , Glucocorticoides/metabolismo , Animales , Células Cultivadas , Cartilla de ADN , Vectores Genéticos/metabolismo , Luciferasas/metabolismo , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Transgenes/genéticaRESUMEN
The LCR/MEL system (Locus Control Region/Murine Erythroleukaemia cells) was employed to express and characterize the Locusta migratoria tyramine receptor (TyrLoc), an insect G protein-coupled receptor. Functional agonist-dependent responses were recorded in stable, tyramine receptor expressing cell clones (MEL-TyrLoc). Tyramine elicited a dose-dependent increase of cytosolic Ca2+-ions and an attenuation of forskolin-induced cyclic adenosine monophosphate (AMP) production. Octopamine was shown to be a weak agonist for both responses. In addition, yohimbine proved to be a potent tyramine receptor antagonist. This study reports the first application of the LCR/MEL expression system in functional assays for G protein-coupled receptors and therefore expands the capabilities of this system by exploiting the functionality of the signal transduction pathways.
Asunto(s)
Expresión Génica , Saltamontes/genética , Receptores de Amina Biogénica/genética , Animales , Secuencia de Bases , Calcio , AMP Cíclico , ADN Complementario , Leucemia Eritroblástica Aguda , Ratones , Datos de Secuencia Molecular , Células Tumorales CultivadasRESUMEN
A cDNA clone of the gene coding for the paralytic neurotoxin (tox34) from the female straw itch mite, Pyemotes tritici, was created by RT-PCR and inserted into the genome of the Autographa californica nucleopolyhedrovirus (AcMNPV) under the control of the AcMNPV p10 promoter. This recombinant virus, AcTOX34.4, caused a rigid paralysis in infected larvae. The infectivity of AcTOX34.4 was compared to the wild-type parent strain, AcMNPV-C6, in second and fourth instar larvae of the cabbage looper, Trichoplusia ni. There were no significant differences in LD(50) values between the recombinant virus and its wild-type parent strain but, as expected, the LD(50) was lower for second instar larvae. The mean time to death and yield of occlusion bodies were measured in second and fourth instar T. ni larvae at a high (100% mortality) and low (<50% mortality) doses of the virus. The mean time to death of recombinant infected larvae was reduced by 50-60% compared to larvae infected with the wild-type strain, depending on virus dose and instar, with these larvae becoming paralysed after approximately 60 h and dying 10-20 h later. This is among the fastest speeds of kill recorded for recombinant baculoviruses. Fourth instar larvae were found to succumb to the recombinant virus more quickly than the second instar larvae. The increase in the speed of kill of the recombinant virus was accompanied by a large reduction of approximately 95% in the yield of progeny virus. The yield of virus showed a highly significant relationship with time to death, but this relationship was complex and varied between the different viruses, concentrations, and instars. The yield per unit weight of the larvae was found to be constant at a low virus dose and increased over time at a high virus dose, irrespective of instar and virus. It is predicted that these changes in the performance of the recombinant virus would act toward reducing its fitness, leading to it being outcompeted by the wild type in field situations.
Asunto(s)
Vectores Genéticos , Ácaros , Nucleopoliedrovirus , Proteínas/genética , Toxinas Biológicas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Conducta Alimentaria , Vectores Genéticos/genética , Vectores Genéticos/fisiología , Larva , Datos de Secuencia Molecular , Mariposas Nocturnas , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/fisiología , Proteínas/fisiología , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Factores de TiempoRESUMEN
Differential screening of two libraries made from whole, immature maize tassels was used to isolate six cDNAs which show enhanced levels of expression in male flowers. MFS1, MFS2, MFS4, MFS10 and MFS18, which were isolated from a 5 cm tassel library, are expressed throughout tassel growth up until mature pollen is produced in the anthers. MFS14, which was isolated from a 10-12 cm tassel library, has a narrower window of expression associated with microsporogenesis and declines as mature pollen is produced. MFS18 mRNA accumulates in the glumes and in anther walls, paleas and lemmas of mature florets. MFS18 mRNA is particularly associated with the vascular bundle in the glumes and encodes a polypeptide of 12 kDa, rich in glycine, proline and serine that has similarities with other plant structural proteins. In contrast, MFS14 mRNA accumulates in the tapetum and encodes a polypeptide of 13 kDa that is rich in alanine. The MFS14 and MFS18 proteins are basic (isoelectric points of 11.56 and 9.54, respectively) and both have hydrophobic N-termini which display all the characteristics of signal peptides, indicating that these proteins may be secreted.
