RESUMEN
The life cycles of sexually reproducing animals and flowering plants begin with male and female gametes and their fusion to form a zygote. Selection at this earliest stage is crucial for offspring quality and raises similar evolutionary issues, yet zoology and botany use dissimilar approaches. There are striking parallels in the role of prezygotic competition for sexual selection on males, cryptic female choice, sexual conflict, and against selfish genetic elements and genetic incompatibility. In both groups, understanding the evolution of sex-specific and reproductive traits will require an appreciation of the effects of prezygotic competition on fitness.
Asunto(s)
Evolución Biológica , Magnoliopsida/fisiología , Polen/fisiología , Reproducción , Conducta Sexual Animal , Espermatozoides/fisiología , Animales , Conducta Competitiva , Copulación , Femenino , Expresión Génica , Masculino , Selección Genética , Caracteres SexualesRESUMEN
Mating affects the reproductive behaviour of insect females: the egg-laying rate increases and courting males are rejected. These post-mating responses are induced mainly by seminal fluid. In Drosophila melanogaster, males transfer two peptides (sex-peptides, = Sps) that reduce receptivity and elicit increased egg laying in their mating partners. Similarities in the open reading frames of the genes suggest that they have arisen by gene duplication. In females, Sps bind to specific sites in the central and peripheral nervous system, and to the genital tract. The binding proteins of the nervous system and genital tract are membrane proteins, but they differ molecularly. The former protein is proposed to be a receptor located at the top of a signalling cascade leading to the two post-mating responses, whereas the latter is a carrier protein moving Sps from the genital tract into the haemolymph. Sps bind to sperm. Together with sperm they are responsible for the persistence of the two post-mating responses. But Sps are the molecular basis of the sperm effect; sperm is merely the carrier.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/fisiología , Péptidos/fisiología , Secuencia de Aminoácidos , Animales , AMP Cíclico/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/genética , Femenino , Genes de Insecto , Péptidos y Proteínas de Señalización Intercelular , Hormonas Juveniles/biosíntesis , Masculino , Modelos Biológicos , Datos de Secuencia Molecular , Familia de Multigenes , Fenómenos Fisiológicos del Sistema Nervioso , Oogénesis , Péptidos/química , Péptidos/genética , Semen/fisiología , Homología de Secuencia de Aminoácido , Conducta Sexual Animal/fisiología , Espermatozoides/fisiologíaRESUMEN
Male insect accessory glands contain factors that are transferred during mating to the female, some inducing post-mating behavior, including the cessation of pheromone production, non-receptivity and the initiation of oviposition. One such factor is the Drosophila melanogaster sex-peptide (DrmSP). A pheromone suppression peptide, termed HezPSP, was identified in the moth Helicoverpa zea, isolated by HPLC and the active peak sequenced, but the activity of the synthesized peptide has not been reported to date. HezPSP bears no sequence homology to DrmSP. However, both peptides contain a disulfide bridge separated by an equal number, but dissimilar, amino acids. We herein report on the pheromonostatic activity of HezPSP partial peptides in the moth Helicoverpa armigera.
Asunto(s)
Proteínas de Insectos/farmacología , Mariposas Nocturnas/efectos de los fármacos , Mariposas Nocturnas/fisiología , Fragmentos de Péptidos/farmacología , Atractivos Sexuales/antagonistas & inhibidores , Atractivos Sexuales/biosíntesis , Conducta Sexual Animal/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Copulación/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Proteínas de Insectos/química , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Alineación de Secuencia , Atractivos Sexuales/química , Atractivos Sexuales/farmacologíaRESUMEN
After mating, Drosophila melanogaster females lay substantially more eggs and mate rarely. Central to these changes is Sex peptide (SP), a male peptide transferred into the female during copulation. Injected into virgins, SP induces the same post mating response as observed after mating. In this study we investigated the role of the mushroom body (MB) in the SP response system. The SP response of females with either chemically ablated or mutant MBs was analyzed. After injection of SP, females with chemically ablated MBs reduce their receptivity and increase their ovulation and oviposition to the level of females with intact MBs. Virgin females with ablated MBs, however, show a constitutively elevated oviposition rate. Hence in untreated females, MBs are not implicated in the SP-induced reduction of receptivity and increase of ovulation. However, they depress the oviposition rate of virgins. Thus, SP has two functions for oviposition: it de-represses the MB-dependent block on the egg laying activity of virgins and additionally stimulates oviposition. SP-injected mushroom body miniature (mbm) females lay fewer eggs, ovulate less frequently, and mate more often than wild-type females. A model of the putative role of MBs and the gene product of mbm in SP-induced oviposition is presented.
Asunto(s)
Copulación/fisiología , Proteínas de Drosophila , Drosophila melanogaster/fisiología , Hormonas de Insectos/fisiología , Cuerpos Pedunculados/fisiología , Péptidos/fisiología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Femenino , Hidroxiurea/farmacología , Péptidos y Proteínas de Señalización Intercelular , Cuerpos Pedunculados/efectos de los fármacos , Oviposición/efectos de los fármacos , Oviposición/fisiología , Ovulación/efectos de los fármacos , Péptidos/farmacología , Fenotipo , Conducta Sexual Animal/fisiologíaRESUMEN
Drosophila melanogaster sex peptide (SP) and Ductus ejaculatorius peptide (DUP99B) are male pheromones transferred in the seminal fluid to the female during copulation. Both reduce sexual receptivity and stimulate oviposition in females. The presence of high-affinity SP and DUP99B binding sites in the female were investigated by incubation of cryostat tissue sections with (125)I-iodinated peptides and subsequent autoradiography. We found that in adult females radiolabeled SP and DUP99B bind to peripheral nerves, the subesophageal ganglion, the cervical connective, to discrete parts of the thoracic ganglion, and to the genital tract. Weak and uniform labeling was detected in the neuropil of the brain and the thoracic ganglion. The labeling pattern in the nervous system suggests binding of the peptides to sensory afferents or glial cells. Scatchard analysis of the binding of (125)I-DUP99B to antennal nerves yielded a dissociation constant K(d) of 6.4 nM. Competition experiments with peptide fragments show that the peptides bind with their homologous C-terminal regions. Binding sites in the nervous system of females are established throughout sexual maturation. Prominent binding of the peptides to afferent nerves suggests modification of sensory input.
Asunto(s)
Células Quimiorreceptoras/metabolismo , Proteínas de Drosophila , Genitales Femeninos/metabolismo , Nervios Periféricos/metabolismo , Atractivos Sexuales/metabolismo , Conducta Sexual Animal/fisiología , Animales , Drosophila melanogaster , Femenino , Péptidos y Proteínas de Señalización Intercelular , Masculino , Péptidos/metabolismoRESUMEN
Sex peptide (SP) and Ductus ejaculatorius peptide (Dup) 99B are synthesized in the retrogonadal complex of adult male Drosophila melanogaster, and are transferred in the male seminal fluid to the female genital tract during mating. They have been sequenced and shown to exhibit a high degree of homology in the C-terminal region. Both affect subsequent mating and oviposition by female D. melanogaster. SP also increases in vitro juvenile hormone (JH) biosynthesis in excised corpora allata (CA) of D. melanogaster and Helicoverpa armigera. We herein report that the partial C-terminal peptides SP(8-36) and SP(21-36) of D. melanogaster, and the truncated N-terminal SP(6-20) do not stimulate JH biosynthesis in vitro in CA of both species. Both of these C-terminal peptides reduce JH-III biosynthesis significantly. Dup99B, with no appreciable homology to SP in the N-terminal region, similarly lacks an effect on JH production by H. armigera CA. In contrast, the N-terminal peptides - SP(1-11) and SP(1-22) - do significantly activate JH biosynthesis of both species in vitro. We conclude that the first five N-terminal amino acid residues at the least, are essential for allatal stimulation in these disparate insect species. We have previously shown that the full-length SP(1-36) depresses pheromone biosynthesis in H. armigera in vivo and in vitro. We now show that full-length Dup99B and the C-terminal partial sequence SP(8-36) at low concentrations strongly depress (in the range of 90% inhibition) PBAN-stimulated pheromone biosynthesis of H. armigera. In addition, the N-terminal peptide SP(1-22), the shorter N-terminal peptide SP(1-11) and the truncated N-terminal SP(6-20) strongly inhibit pheromone biosynthesis at higher concentrations.
Asunto(s)
Drosophila melanogaster/fisiología , Hormonas Esteroides Gonadales/fisiología , Mariposas Nocturnas/fisiología , Péptidos/fisiología , Secuencia de Aminoácidos , Animales , Femenino , Hormonas Esteroides Gonadales/biosíntesis , Hormonas Esteroides Gonadales/química , Hormonas Juveniles/biosíntesis , Masculino , Datos de Secuencia Molecular , Péptidos/química , Reproducción/fisiologíaRESUMEN
In many sexually mature insects egg production and oviposition are tightly coupled to copulation. Sex-Peptide is a 36-amino-acid peptide synthesized in the accessory glands of Drosophila melanogaster males and transferred to the female during copulation. Sex-Peptide stimulates vitellogenic oocyte progression through a putative control point at about stage 9 of oogenesis. Here we show that application of the juvenile hormone analogue methoprene mimics the Sex-Peptide-mediated stimulation of vitellogenic oocyte progression in sexually mature virgin females. Apoptosis is induced by 20-hydroxyecdysone in nurse cells of stage 9 egg chambers at physiological concentrations (10(-7) M). 20-Hydroxyecdysone thus acts as an antagonist of early vitellogenic oocyte development. Simultaneous application of juvenile hormone analogue, however, protects early vitellogenic oocytes from 20-hydroxyecdysone-induced resorption. These results suggest that the balance of these hormones in the hemolymph regulates whether oocytes will progress through the control point at stage 9 or undergo apoptosis. These data are further supported by a molecular analysis of the regulation of yolk protein synthesis and uptake into the ovary by the two hormones. We conclude that juvenile hormone is a downstream component in the Sex-Peptide response cascade and acts by stimulating vitellogenic oocyte progression and inhibiting apoptosis. Since juvenile hormone analogue does not elicit increased oviposition and reduced receptivity, Sex-Peptide must have an additional, separate effect on these two postmating responses.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/citología , Ecdisterona/farmacología , Metopreno/farmacología , Péptidos/farmacología , Vitelogénesis/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Copulación , Relación Dosis-Respuesta a Droga , Proteínas del Huevo/biosíntesis , Proteínas del Huevo/genética , Femenino , Hemolinfa/química , Péptidos y Proteínas de Señalización Intercelular , Modelos Biológicos , Imitación Molecular , Oviposición/efectos de los fármacos , ARN Mensajero/análisis , Transducción de SeñalRESUMEN
Previous studies demonstrate that virgin female adult Helicoverpa armigera (Lepidoptera: Noctuidae) moths exhibit calling behaviour and produce sex pheromone in scotophase from the day after emergence, and that mating turns off both of these pre-mating activities. In the fruit fly Drosophila melanogaster, a product of the male accessory glands, termed sex peptide (SP), has been identified as being responsible for suppressing female receptivity after transfer to the female genital tract during mating. Juvenile hormone (JH) production is activated in the D. melanogaster corpus allatum (CA) by SP in vitro. We herein demonstrate cross-reactivity of D. melanogaster SP in the H. armigera moth: JH production in photophase virgin female moth CA in vitro is directly activated in a dose-dependent manner by synthetic D. melanogaster SP, and concurrently inhibits pheromone biosynthesis activating neuropeptide (PBAN)-activated pheromone production by isolated pheromone glands of virgin females. Control peptides (locust adipokinetic hormone, AKH-I, and human corticotropin, ACTH) do not inhibit in vitro pheromone biosynthesis. Moreover, SP injected into virgin H. armigera females, decapitated 24 h after eclosion, or into scotophase virgin females, suppresses pheromone production. In the light of these results, we hypothesize the presumptive existence of a SP-like factor among the peptides transmitted to female H. armigera during copulation, inducing an increased level of JH production and depressing the levels of pheromone produced thereafter.
RESUMEN
Mating elicits two reactions in many insect females: egg deposition is increased and receptivity to males is reduced. Central to the control of receptivity and oviposition in Drosophila melanogaster is the sex peptide (SP), a 36-amino-acid peptide sex pheromone synthesized in the male accessory glands and transferred to the female during copulation. To identify regulatory mechanisms involved in the maintenance of the oviposition response, we have compared the effects of mating and SP application with respect to oogenesis. The distribution of the various stages of oogenesis in the ovary, yolk protein (YP) synthesis by the fat body, as well as YP content, uptake and synthesis by the ovary were investigated. Transcripts of the yolk protein genes (yp) were quantified by Northern blotting. Based on our results, we conclude that mating and SP injection into virgin females stimulate yp gene transcription in the fat body only moderately above the background level. However, uptake into the ovary and transcription of the yp genes in the ovary is strongly enhanced after either mating or SP injection. These data are supported by the finding that the abundance of the vitellogenic stage 10 oocytes is also increased. In contrast, early vitellogenic stages 8 and 9 of oogenesis are present in the same numbers in virgin, mated, and SP-injected females, which suggests a control point at about stage 9 determining vitellogenic oocyte progression. The finding that SP can elicit equally all changes observed after copulation suggests that in the sexually mature female it is the major component controlling and stimulating oogenesis after mating.
Asunto(s)
Yema de Huevo/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Péptidos/farmacología , Atractivos Sexuales/farmacología , Conducta Sexual Animal/fisiología , Animales , Drosophila melanogaster , Proteínas del Huevo/genética , Proteínas del Huevo/metabolismo , Cuerpo Adiposo/metabolismo , Femenino , Hemolinfa/efectos de los fármacos , Hemolinfa/metabolismo , Metionina , Microinyecciones , Ovario/efectos de los fármacos , Ovario/metabolismo , Oviposición , Péptidos/administración & dosificación , Procesamiento Postranscripcional del ARN , Atractivos Sexuales/administración & dosificación , Radioisótopos de Azufre , Transcripción GenéticaRESUMEN
Mating elicits two well-defined reactions in sexually matured females of many insects: reduction of receptivity and increased oviposition. These post-mating responses have been shown to be induced by factors synthesized in the reproductive tract of the adult male and transferred in the seminal fluid to the female during copulation. One of these factors, named sex-peptide (SP), has been identified in Drosophila melanogaster. Using an in vitro radiochemical assay, we show that synthetic sex-peptide considerably activates juvenile hormone III-bisepoxide (JHB3) synthesis in corpus allatum (CA) excised from Days 3 and 4 post-eclosion virgin females. Base levels are significantly lower at emergence (Day 0) than on subsequent days, and only weak stimulation is obtained on Day 1, while none is obtained on Day 2, where maximal basal synthesis occurs. The CA of mated females cannot be stimulated further for at least 7 days, but regain responsiveness by Day 10 after mating. Synthesis of JHB3 stimulated by SP in vitro persists for at least 4 h after removal of the peptide. Development of responsiveness of the CA to SP in vitro is compared with development of the post-mating reactions of sex-peptide injected virgin females. Our results suggest that the CA is a direct target for SP in vivo and that sexual maturity is established separately for the two post-mating reactions.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Biosíntesis de Péptidos , Péptidos , Animales , Corpora Allata/metabolismo , Ácidos Grasos Monoinsaturados/metabolismo , Femenino , Péptidos y Proteínas de Señalización Intercelular , MasculinoRESUMEN
A peptide homologous to the Drosophila melanogaster sex-peptide (SP) was isolated from Drosophila suzukii accessory glands and its amino acid sequence determined. The D. suzukii peptide contains 41 amino acids and has a calculated molecular weight of 5100 Da. Comparison of the sequences reveals strong homologies in the N-terminal and C-terminal parts of the peptides. In the D. suzukii sex-peptide, however, five additional amino acids are inserted after amino acid 7. Based on the sequence of the peptide, a cDNA coding for the D. suzukii peptide was isolated by PCR. Sequence analysis of the cDNA confirmed the SP amino acid sequence determined by peptide sequencing. Furthermore, based on the cDNA sequence, we isolated the D. suzukii sex-peptide gene by inverse PCR. The D. suzukii sex-peptide gene contains an intron and codes for a 60 amino acid precursor. The D. melanogaster and the D. suzuki sex-peptides elicit rejection behaviour in the presence of males and an increased egg laying in virgin females of both species.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila/genética , Hormonas de Insectos/genética , Péptidos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Cromatografía Líquida de Alta Presión , ADN , Femenino , Hormonas de Insectos/fisiología , Péptidos y Proteínas de Señalización Intercelular , Intrones , Datos de Secuencia Molecular , Ovulación , Péptidos/fisiología , Reacción en Cadena de la Polimerasa , Precursores de Proteínas/genética , Especificidad de la Especie , Relación Estructura-ActividadRESUMEN
Injection of a peptide of 36 amino acids into virgin Drosophila females changes their reproductive properties drastically: males are rejected and egg laying is increased. The neuronal and physiological properties of the virgin state are replaced by a new pattern of behavior and stimulation of egg production and deposition. Under natural conditions, the peptide is synthesized by the male and transferred into the female during copulation. The sex-peptide, therefore, can be considered as a pheromone. In this review, I shall limit my discussion to Drosophila melanogaster.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/fisiología , Péptidos/fisiología , Conducta Sexual Animal/fisiología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Copulación/efectos de los fármacos , Copulación/fisiología , Drosophila melanogaster/ultraestructura , Femenino , Genitales Masculinos/metabolismo , Genitales Masculinos/ultraestructura , Insectos/fisiología , Péptidos y Proteínas de Señalización Intercelular , Masculino , Datos de Secuencia Molecular , Oviposición/efectos de los fármacos , Oviposición/fisiología , Péptidos/farmacología , Proteínas Recombinantes de Fusión/biosíntesis , Conducta Sexual Animal/efectos de los fármacos , Especificidad de la EspecieRESUMEN
Sex peptide, a secreted component of the male accessory glands, has been shown to induce behavioral and physiological changes in mated Drosophila. We transformed flies with a hybrid gene containing an hsp70 promoter fused to a cDNA encoding sex peptide. Heat-induced ectopic expression of the peptide in transgenic virgin females altered their reproductive behavior, in the presence of courting males, to that observed in mated females. This demonstrates that the peptide is functional as expected. Time course studies revealed that the behavioral change appeared earlier than the stimulated ovulation. We have also introduced a modified sex peptide gene that is driven by the yp1 enhancer, conferring expression in adult females, and shown that these flies refuse mating constitutively in the presence of courting males and lay unfertilized eggs at the rate of mated females.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/fisiología , Péptidos/metabolismo , Conducta Sexual Animal/fisiología , Animales , Animales Modificados Genéticamente/fisiología , Dípteros/genética , Dípteros/fisiología , Femenino , Calor , Péptidos y Proteínas de Señalización Intercelular , Ovulación/fisiología , Caracteres Sexuales , Distribución TisularRESUMEN
Six tRNA(Leu) isoacceptors from yellow lupin seeds were purified, sequenced, and their readthrough properties over the UAG stop codon were tested using TMV RNA as a messenger. The tested tRNAs(Leu) did not show amber suppressor activity. The partial structure of tRNA(Gln), a minor species in yellow lupin, was also determined. Comparison of the nucleotide sequence of all known isoacceptors of tRNA(Tyr), tRNA(Gln) and tRNA(Leu) from plants, mammals and ciliates enabled us to find general structural requirements for tRNA to be a UAG suppressor. From the partial sequence of lupin tRNA(Gln) we suggest that it will have readthrough properties.
Asunto(s)
Plantas/genética , ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia de Leucina/genética , Supresión Genética , Autorradiografía , Secuencia de Bases , Cromatografía DEAE-Celulosa , Codón , Datos de Secuencia Molecular , Biosíntesis de Proteínas , ARN de Transferencia de Leucina/aislamiento & purificación , Semillas/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Six Drosophila melanogaster tRNA(Tyr) genes have been isolated and sequenced. They contained introns of different sequences and two size classes: 20 or 21 base pairs (bp) (five genes) and 113 bp (one gene). However, the sequences coding for the mature tRNA(Tyr) were identical in all six genes. The 113-bp intron-containing gene was a single-copy gene. Hence, its primary transcript could be traced by S1 mapping. The gene was turned on during embryogenesis and continually expressed to various degrees during the following developmental stages. Thus, S1 mapping is a feasible method to follow the transcriptional activity of individual genes with identical mature products, provided that their primary transcripts are unique. The six genes were organized in two clusters of three and two genes, respectively (each containing a 20- or a 21-bp intron; cytological localization, 85A), and a single-copy gene (113-bp intron; cytological localization, 28C). We show that four of the six tRNA(Tyr) genes characterized were localized in putative 5' control regions of developmentally controlled genes transcribed by polymerase II.
Asunto(s)
Mapeo Cromosómico , Drosophila melanogaster/genética , Genes , ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia de Tirosina/genética , Transcripción Genética , Animales , Secuencia de Bases , Intrones , Datos de Secuencia Molecular , Hibridación de Ácido NucleicoRESUMEN
In Saccharomyces cerevisiae, pseudouridine formation in the middle position of the tRNA(Tyr) anticodon (psi 35) is dependent on the presence of the intron in the tRNA(Tyr) gene (Johnson and Abelson, Nature 302:681-687, 1983). Drosophila melanogaster tRNA(Tyr) genes contain introns of three size classes: 20 or 21 base pairs (bp) (six genes), 48 bp (one gene), and 113 bp (one gene). As in yeast, removal of the intron led to loss of psi 35 in the anticodon when transcription was assayed in Xenopus laevis oocytes. All Drosophila intron sizes supported psi 35 formation. The same results were obtained with the homologous X. laevis tRNA(Tyr) genes containing introns of 12 or 13 bp or with a deleted intron. The introns of yeast (Nishikura and DeRobertis, J. Mol. Biol. 145:405-420, 1981), D. melanogaster, and X. laevis tRNA(Tyr) wild-type genes, while they all supported psi 35 synthesis, did not share any consensus sequences. As discussed, these results, taken together, suggest that for appropriate function the psi 35 enzyme in the X. laevis oocyte needs the presence of an unqualified intron in the tRNA gene and a tRNA(Tyr)-like structure in the unprocessed tRNA precursor.
Asunto(s)
Anticodón , Genes , Intrones , Seudouridina , ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia de Tirosina/genética , ARN de Transferencia , Uridina , Animales , Secuencia de Bases , Drosophila melanogaster/genética , Femenino , Datos de Secuencia Molecular , Mutación , Oocitos/enzimología , Uridina/análogos & derivados , Xenopus laevisRESUMEN
Seven xanthine dehydrogenase and cross-reacting material negative Drosophila melanogaster rosy stocks were screened for amber and ochre nonsense mutations. Amber and ochre nonsense suppressors were created by site-directed mutagenesis starting from a wild-type tRNA(Tyr) gene. The suppressor tRNA genes were subcloned into a pUChsneo transformation vector providing heat-shock controlled neomycin resistance. The seven rosy stocks were germline transformed with amber and ochre tDNA(Tyr), and the G1 generation was screened for Geneticin resistance. Surviving rosy516 flies transformed with the amber suppressor showed an eye colour intermediate between the original ry516 stock and the wild-type, suggesting that ry516 is an amber nonsense mutant. This was confirmed by sequencing the relevant part of the ry516 gene; the analysis revealed a C-to-T transition in a CAG glutamine codon at nucleotide 1522 of the wild-type rosy gene.
Asunto(s)
ARN de Transferencia/genética , Supresión Genética , Transformación Genética , Animales , Northern Blotting , Clonación Molecular , Drosophila melanogaster , Femenino , Masculino , Mutación , Transcripción GenéticaRESUMEN
The nucleotide sequence of the two major Drosophila melanogaster tRNATyr isoacceptors was determined to be pC-C-U-U-C-G-A-U-A-m2G-C-U-C-A-G-D-D-G-G-acp3 U-A-G-A-G-C-m2(2)G-G-psi-G-G-A-C-U-G/Q-psi-A-m1G-A-Um-C-C-A-U-A-G-m7 G-D-C-G-C-U-G-G-U(T)-psi-C-A-m1A-A-U-C-C-G-G-C-U-C-G-A-A-G-G-A-A-C-C-AOH . The two isoacceptors differ by the presence of a G or a Q in the wobble position. Both contain a partial modification in position 54 (U/T). Thus, these tRNAs are transcribed from a single gene (or many genes with identical sequences). A fast and sensitive postlabeling method for sequencing tRNA anticodons is described. Nuclease S-1-treated tRNA is labeled with 5[32P]-pCp using T-4 RNA ligase. The tRNA fragments are then separated on 7 M urea/20% PAA gels. After autoradiography the RNA is eluted and digested with T-2 RNase. The nature of the labeled nucleotides is determined by two-dimensional thin-layer chromatography. The same method can be used to determine the 5' sequence of a tRNA by 3' labeling 5' tRNA halves with 5[32P]-pCp and subsequent chemical sequencing.