Ectopic expression of sex peptide alters reproductive behavior of female D. melanogaster.

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.

tRNA(Tyr) genes of Drosophila melanogaster: expression of single-copy genes studied by S1 mapping.

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.

Pseudouridine modification in the tRNA(Tyr) anticodon is dependent on the presence, but independent of the size and sequence, of the intron in eucaryotic tRNA(Tyr) genes.

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.

The primary structure of six leucine isoacceptor tRNAs of yellow lupin seeds. The structural requirements for amber tRNA suppressor activity.

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.

Transfer RNA in aging Drosophila: I. Extent of aminoacylation.

Transfer RNA and aminoacyl-tRNA synthetases have been isolated from 5 day and 35 day old Drosophila melanogaster males. Transfer RNA isolated from old males cannot be aminoacylated to the same extent as tRNA from young flies. The average reduction is 16% and 53% for tRNALeu. The aminoacylation ability of some aminoacyl-tRNA synthetases isolated from 35 day old flies is reduced by more than 50%.

Transfer RNA in aging Drosophila: II. Isoacceptor patterns.

Transfer RNA and aminoacyl-tRNA synthetases have been isolated from 5 and 35 day old Drosophila melanogaster males. The isoacceptor pattern of the following tRNAs is not changed irrespective of the source of the tRNA and amino-acylating enzymes: tRNAAla, tRNALeu, tRNAMet, and tRNASer. However, the Q base containing tRNAAsp, tRNAAsN, tRNAHis, and tRNATyr show dramatic changes in their isoacceptor patterns during the aging process. This probably reflects a change in the activity of a tRNA modifying enzyme. These findings are discussed in relation to Strehler's theory of aging and development.

The primary structure of wheat germ tRNAArg--the substrate for arginyl-tRNAArg:protein transferase.

Besides its major role in protein synthesis, wheat germ arginyl-tRNAArg can serve as an amino acid donor in an enzymatic reaction to bovine serum albumin catalysed by the enzyme arginyl-tRNAArg: protein transferase. The nucleotide sequence of the tRNAArg involved in this reaction was determined to be: pG-A-C-U-C-C-G-U-m1G-m2G-C-C-C-A-A-D-Gm-G-A-X-A-A-G-G-C-m2(2) G-C-U-G-G-U-Cm-U-I-C-G-m2A-A-A-C-C-A-G-A-G-A-D-U-m5C-U-G-G-G-T-psi -C-G-m1 A-U-C-C-C-C-A-G-C-G-G-A-G-U-C-G-C-C-AOH. We suggest that the decapentanucleotide 5'-G-U-Pu-m2G-C-N-C-A-A-D-Gm-G-A-X-A-3', localized in the D-region, interacts specifically with the protein transferase.

Drosophila suzukii contains a peptide homologous to the Drosophila melanogaster sex-peptide and functional in both species.

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.

Common functional elements of Drosophila melanogaster seminal peptides involved in reproduction of Drosophila melanogaster and Helicoverpa armigera females.

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.

Inhibition of pheromone biosynthesis in Helicoverpa armigera by pheromonostatic peptides.

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.

The sex-peptide.

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.

Sex-peptides: seminal peptides of the Drosophila male.

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.

The structure and function of tRNA genes of higher eukaryotes.

The most recent findings concerning the structure and function of tRNA genes of higher eukaryotes are discussed in an exemplary way. The tRNA genes of higher organisms are either dispersed or clustered at different sites of the genome. Clusters contain tRNA genes oriented in both directions and on both strands of the DNA with spacers of various length inbetween. Some genes contain intervening sequences close to the 3' side of the anticodon. The primary transcription product possesses a 5' leader and a 3' trailer sequence which are removed by several maturation steps in a strict temporal and spacial order. Internal transcription control regions (promotors) are located at the 5' and 3' ends of the mature tRNA coding section of the tRNA gene. External sequences modulating the efficiency of the expression are present at the immediate 5' ends of the genes. Transfer RNA genes are located nonrandomly in the nucleosomes.

The nucleotide sequence of phenylalanine tRNA2 of Drosophila melanogaster: four isoacceptors with one basic sequence.

The nucleotide sequence of Drosophila melanogaster phenylalanine tRNA2 was determined to be: pG-C-C-G-A-A-A-U-A-M2G-C-U-C-A-G-D-D-G-G-G-A-G-A-G-C-m22G-psi-psi-A-G-A-C(m)-U-Gm-A-A-mlG-A-psi-C-U-A-A-A-G-m7G-U(D)-C-C-C-C-G-G-T-psi-C-A-mlA-U-C-C-G-G-G-U-U-U-C-G-G-C-A-C-C-AOH. Upon RPC-5 chromatography at pH 3.8 tRNA2Phe can be separated into four isoacceptors due to the partial modifications in positions 32 and 47. Thus the posttranscriptional modification of tRNA2Phe transcribed from one gene (or many genes with identical sequences results in four isoacceptors with the same basic sequence.