The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila.

The initiation factor 4E for eukaryotic translation (eIF4E) binds the messenger RNA 5'-cap structure and is important in the regulation of protein synthesis. Mammalian eIF4E activity is inhibited when the initiation factor binds to the translational repressors, the 4E-binding proteins (4E-BPS). Here we show that the Drosophila melanogaster 4E-BP (d4E-BP) is a downstream target of the phosphatidylinositol-3-OH kinase (PI(3)K) signal-transduction cascade, which affects the interaction of d4E-BP with eIF4E. Ectopic expression of a highly active d4E-BP mutant in wing-imaginal discs causes a reduction of wing size, brought about by a decrease in cell size and number. A marked reduction in cell size was also observed in post-mitotic cells. Expression of d4E-BP in the eye and wing together with PI(3)K or dAkt1, the serine/threonine kinase downstream of PI(3)K, resulted in suppression of the growth phenotype elicited by these kinases. Our results support a role for d4E-BP as an effector of cell growth.

Requirement for a noncoding RNA in Drosophila polar granules for germ cell establishment.

In Drosophila embryos, germ cell formation is induced by specialized cytoplasm at the posterior of the egg, the pole plasm. Pole plasm contains polar granules, organelles in which maternally produced molecules required for germ cell formation are assembled. An untranslatable RNA, called Polar granule component (Pgc), was identified and found to be localized in polar granules. Most pole cells in embryos produced by transgenic females expressing antisense Pgc RNA failed to complete migration and to populate the embryonic gonads, and females that developed from these embryos often had agametic ovaries. These results support an essential role for Pgc RNA in germline development.

A Drosophila melanogaster homologue of the human DEAD-box gene DDX1.

DEAD-box genes are found throughout evolution and encode RNA-binding proteins. Such proteins include eukaryotic initiation factor-4A, which is essential for protein translation, Vasa, which is essential for germ line development, and a number of nuclear and mitochondrial RNA splicing factors. Transcription of a human DEAD-box gene, DDX1, is elevated in two retinoblastoma cell lines as a result of amplification of the immediate chromosomal region surrounding it, suggesting an important role for this gene in control of cell growth and division. We have isolated a Drosophila melanogaster (Dm) homologue (Ddx1) of DDX1 which is strikingly similar to the human gene. The similarity (58.3% amino acid (aa) identity over 720 aa) extends beyond regions conserved in all DEAD-box proteins and covers the entire lengths of the proteins. The 2.7-kb Dm Ddx1 RNA is expressed throughout development, but its levels are elevated in early embryos. Ddx1 maps to polytene chromosome band 79D4 on the left arm of Dm chromosome 3.

Localized Bicaudal-C RNA encodes a protein containing a KH domain, the RNA binding motif of FMR1.

The Bicaudal-C (Bic-C) gene of Drosophila melanogaster is required for correct targeting of the migrating anterior follicle cells and for specifying anterior position. Females lacking any wild type copies of Bic-C produce only eggshells open at the anterior end, because of the failure of the columnar follicle cells to migrate in the correct position at the nurse cell--oocyte boundary. Embryos which develop from eggs produced in females with only one wild type copy of Bic-C show defects in anterior patterning and an abnormal persistence of oskar RNA in anterior regions. We cloned Bic-C and found that, in ovaries, Bic-C RNA is expressed only in germline cells. Bic-C RNA is localized to the oocyte in early oogenesis, and later concentrates at its anterior cortex. The Bic-C protein includes five KH domains similar to those found in the human fragile-X protein FMR1. Alteration of a highly conserved KH domain codon by mutation abrogates in vivo Bic-C function. These results suggest roles for the Bic-C protein in localizing RNAs and in intercellular signaling.

Dbp45A encodes a Drosophila DEAD box protein with similarity to a putative yeast helicase involved in ribosome assembly.

Proteins of the DEAD family of putative ATP-dependent RNA helicases have been implicated in translation initiation, ribosome assembly, and RNA processing in a variety of organisms from Escherichia coli to man. Among these proteins are eIF-4A, an essential component of the cap-binding complex, numerous yeast proteins required for pre-mRNA splicing, and proteins from yeast and E. coli necessary for ribosome assembly. We report the isolation of a new DEAD gene from Drosophila, Dbp45A, which is most abundantly expressed in 6-12 h embryos and adults. The predicted amino acid sequence of the Dbp45A product contains all eight highly conserved DEAD family motifs, and most closely resembles the Saccharomyces cerevisiae DRS1p among known DEAD box proteins. DRS1p has been implicated in ribosomal RNA processing.

Molecular movements in oocyte patterning and pole cell differentiation.

Central to the differentiation and patterning of the Drosophila oocyte is the asymmetric intracellular localization of numerous mRNA and protein molecules involved in developmental signalling. Recent advances have identified some of the molecules mediating oocyte differentiation, specification of the anterior pole of the embryo, and determination of the embryonic germ line. This work is considered in the context of the classical model of the germ plasm as a cytoplasmic determinant for germ cell formation.

Dbp73D, a Drosophila gene expressed in ovary, encodes a novel D-E-A-D box protein.

Proteins of the D-E-A-D family of putative ATP-dependent RNA helicases have been implicated in translation initiation and RNA splicing in a variety of organisms from E. coli to man. The Drosophila vasa protein, a member of this family, is required in the female germ line for fertility and for specification of germ line and posterior positional information in progeny embryos. We report the isolation of another D-E-A-D gene from Drosophila, which, like vasa, is expressed in germ line tissue. The predicted amino acid sequence of this new gene, Dbp73D, contains all of the highly conserved helicase motifs, but is otherwise the farthest-diverged member of the family so far identified.

The genetics of a small autosomal region of Drosophila melanogaster containing the structural gene for alcohol dehydrogenase. VII. Characterization of the region around the snail and cactus loci.

The genetic interval 35C to 36A on chromosome arm 2L of Drosophila melanogaster has been saturated for mutations with visible or lethal phenotypes. 38 loci have been characterized, including several maternal-effect lethals (vasa, Bic-C, chiffon, cactus and cornichon) and several early embryonic lethals, including snail and fizzy. About 130 deletions have been used to order these loci. Complex interactions between mutant alleles have been uncovered in the immediate genetic environs of the snail gene, as has further evidence for an interaction between this region and that including the nearby genes no-ocelli and elbow.

Posterior localization of vasa protein correlates with, but is not sufficient for, pole cell development.

The protein product of the Drosophila maternal-effect posterior group gene vasa is localized to the posterior pole of the oocyte and is sequestered by the pole cells as they form. It is, however, present at easily detectable levels throughout the oocyte and pre-blastoderm embryo. The protein is present in the pole cells and their germ line derivatives throughout all stages of development. An antiserum against this protein recognizes a pole-cell-specific antigen in seven other Drosophila species. Of six other maternal-effect loci essential for embryonic pole cell development, none affects expression of vasa, mutations in four abolish vasa protein localization, and mutations in two, tudor and valois, have little, if any, effect on vasa expression or localization. This indicates that vasa protein, when properly localized, is not sufficient for induction of pole cell development, and that at least the tudor and valois wild-type functions are also required specifically for this process. These results are discussed with respect to the multiple functions of the vasa gene.

Homeosis and the interaction of zeste and white in Drosophila.

Transvection effects in Drosophila melanogaster suggest a form of gene modulation that is responsive to the proximity of homologous genes. These effects have been well characterized at bithorax and decapentaplegic, and in the interaction between the zeste and white genes. The mechanistic basis for transvection is not known. As part of a genetic analysis of transvection, a study is being made of a class of mutations defined as modifiers of the eye color resulting from the interaction of zeste and white. This report details the observations that several of these mutations also have homeotic effects.

The product of the Drosophila gene vasa is very similar to eukaryotic initiation factor-4A.

The vasa gene product of Drosophila melanogaster is required only in the female germ line. Progeny of females homozygous for vasa mutations lack posterior structures and pole cells. Isolation and characterization of vasa genomic and complementary DNA clones show that the transcript is abundant in the female germ line and early embryos only. The predicted amino acid sequence is very similar to those of the translation initiation factor eIF-4A and the human nuclear antigen p68.

Studies of the genetic organization of the vestigial microregion of Drosophila melanogaster.

The region of the second chromosome of Drosophila melanogaster defined by Df(2R)vgB was screened for recessive lethal and visible mutations. Fifty-eight new recessive alleles fall into 17 complementation groups. Many new vg alleles were also isolated in a screen for new vg deficiencies. The breakpoints of the new vg deficiencies were nonrandomly distributed. The distal breakpoints of twelve of 20 deficiencies overlapping Df(2R)vgB are genetically identical to that of Df(2R)vgD, coinciding with the position of a complex, pleiotropic locus, l(2)49Ea-Psc-Su(z)2.

Proline transport in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is capable of utilizing proline as the sole source of nitrogen. Mutants of S. cerevisiae with defective proline transport were isolated by selecting for resistance to either of the toxic proline analogs L-azetidine-2-carboxylate or 3,4-dehydro-DL-proline. Strains carrying the put4 mutation are defective in the high-affinity proline transport system. These mutants could still grow when given high concentrations of proline, due to the operation of low-affinity systems whose existence as confirmed by kinetic studies. Both systems were repressed by ammonium ions, and either was induce by proline. Low-affinity transport was inhibited by histidine, so put4 mutants were unable to grow on a medium containing high concentrations of proline to which histidine has been added.