Mutations in the predicted aspartyl tRNA synthetase of Drosophila are lethal and function as dosage-sensitive maternal modifiers of the sex determination gene Sex-lethal.

Stable activation of the Drosophila sex determination gene Sex-lethal in the female embryo is a multistep process. Early in embryogenesis Sex-lethal is regulated at the level of transcription, and then later in embryogenesis Sex-lethal regulation switches to an autoregulatory RNA splicing mechanism. Previous studies have shown that successful activation of Sxl requires both maternally and zygotically provided gene products, many of which are essential for viability and have other, non-sex specific functions. Using a screen for dosage-sensitive modifiers we identified a new maternally expressed gene, l(2)49Db, as a likely participant in Sxl activation. We show that the establishment of the Sxl autoregulatory splicing loop, but not the earlier steps in Sxl activation, is sensitive to the maternal dosage of l(2)49Db. We further demonstrate that l(2)49Db encodes an aspartyl tRNA synthetase. Finally we present evidence that this effect is indirect, by demonstrating that mutations in tryptophanyl tRNA synthetase are also dosage-sensitive maternal modifiers of Sex-lethal. These data suggest that stable activation of Sex-lethal in the embryo may be particularly sensitive to perturbation of the translational machinery.

The function of the Drosophila thioredoxin homologue encoded by the deadhead gene is redox-dependent and blocks the initiation of development but not DNA synthesis.

Thioredoxin is a highly conserved disulfide reducing protein whose structure and biochemical properties have been extensively studied. Nonetheless, its function in vivo is not well defined. In Drosophila, the maternal-effect gene deadhead encodes a thioredoxin-like protein that is required for initiation of embryonic development. Here we report that deadhead function is dependent on its enzymatic activity: transgenes carrying mutations in thioredoxin's conserved active site failed to rescue the deadhead mutant phenotype. A number of studies have documented that thioredoxin plays a role in DNA synthesis. If thioredoxin is required for DNA synthesis in the fly, then deadhead mutations will suppress mutations that inappropriately synthesize DNA. Contrary to expectation, we find that deadhead does not function as a suppressor in this assay. The observed epistatic relationship between these mutations clearly indicates that deadhead is not essential for DNA metabolism. The possibility of a regulatory role in controlling the initiation of S-phase is discussed.

Immunologic responses to gene transfer into mice via the polymeric immunoglobulin receptor.

The respiratory epithelium is the primary target tissue for gene therapy of cystic fibrosis, and several methods of gene transfer permit the introduction of the gene encoding the normal cystic fibrosis transmembrane conductance regulator into cells of the respiratory tract in animals. DNA complexes based on Fab antibodies to secretory component have been used to mediate the delivery and uptake of expression plasmids into the respiratory tract via the polymeric immunoglobulin receptor both in vitro and in vivo. We evaluated the efficacy of gene transfer after several administrations of the DNA complexes, and examined the immunogenicity and toxicity of repetitive administration of anti-secretory component Fab-based complexes. Mice received single or multiple injections of the DNA complexes containing the plasmid pGL2 every 21 days after the initial treatment, and lysates from the lung and liver were assayed for luciferase expression. Luciferase activity was detected in the lungs of mice that received a single injection of the DNA complexes, whereas transgene expression was significantly lower in the mice that received three injections of the DNA complexes (17338 +/- 5469 integrated light units/mg and 3771 +/- 1778 integrated light units/mg, respectively). Serum samples from animals that underwent single or multiple injections were analyzed for a serologic response against the conjugate-DNA complexes by ELISA. No anticomplex antibodies were detected in the mice after a single injection. An escalating antibody response was noted with increasing number of treatments with the conjugate-DNA complexes. This serologic response was directed exclusively against the rabbit-derived, anti-secretory component (anti-SC) Fab antibody, and not against either the plasmid DNA or poly-L-lysine. Single injection of the conjugate-DNA complexes did not result in the consumption of circulating complement. Using direct immunofluorescence, perivascular deposits of immunoglobulin G were found in the liver of animals that received three treatments; no such deposition was detected in the lungs or kidneys. No increase in inflammatory cell infiltrates was observed in tissues after single and repeated injections of the DNA complexes. Thus, we conclude that repeated injections of the anti-SC Fab-based complexes evoked a humoral immune response against the heterologous Fab portion of the complex that was associated with reduced efficiency of gene transfer.

The putative Drosophila NMDARA1 gene is located on the second chromosome and is ubiquitously expressed in embryogenesis.

We describe the cDNA sequence of a Drosophila gene located at 49D on the second chromosome. The predicted protein sequence has extensive homology (46% identity, 76% similarity) along its entire length to the central portion of the mammalian brain-specific NMDARA1 glutamate binding protein. While RNA expression studies of the Drosophila protein shown that it is ubiquitously expressed throughout embryogenesis, there appears to be a selective accumulation of transcript in the precursor for the larval brain. The similarity in both expression and sequence suggests that the two proteins may share a conserved function.

A late Golgi sorting function for Saccharomyces cerevisiae Apm1p, but not for Apm2p, a second yeast clathrin AP medium chain-related protein.

Mammalian clathrin-associated protein (AP) complexes, AP-1 (trans-Golgi network) and AP-2 (plasma membrane), are composed of two large subunits of 91-107 kDa, one medium chain (mu) of 47-50 kDa and one small chain (sigma) of 17-19 kDa. Two yeast genes, APM1 and APM2, have been identified that encode proteins related to AP mu chains. APM1, whose sequence was reported previously, codes for a protein of 54 kDa that has greatest similarity to the mammalian 47-kDa mu 1 chain of AP-1. APM2 encodes an AP medium chain-related protein of 605 amino acids (predicted molecular weight of 70 kDa) that is only 30-33% identical to the other family members. In yeast containing a normal clathrin heavy chain gene (CHC1), disruptions of the APM genes, singly or in combination, had no detectable phenotypic consequences. However, deletion of APM1 greatly enhanced the temperature-sensitive growth phenotype and the alpha-factor processing defect displayed by cells carrying a temperature-sensitive allele of the clathrin heavy chain gene. In contrast, deletion of APM2 caused no synthetic phenotypes with clathrin mutants. Biochemical analysis indicated that Apm1p and Apm2p are components of distinct high molecular weight complexes. Apm1p, Apm2p, and clathrin cofractionated in a discrete vesicle population, and the association of Apm1p with the vesicles was disrupted in CHC1 deletion strains. These results suggest that Apm1p is a component of an AP-1-like complex that participates with clathrin in sorting at the trans-Golgi in yeast. We propose that Apm2p represents a new class of AP-medium chain-related proteins that may be involved in a nonclathrin-mediated vesicular transport process in eukaryotic cells.

The Drosophila maternal effect locus deadhead encodes a thioredoxin homolog required for female meiosis and early embryonic development.

This study describes the identification, function and molecular characterization of deadhead, a Drosophila thioredoxin homolog. Although in vitro studies have shown that thioredoxin can post-translationally regulate the activity of many different proteins, we find that this homolog is not essential for viability. The phenotypic analysis of two different mutations which eliminate function suggests that dhd is essential for female meiosis. The majority of eggs laid by females homozygous for null mutations are fertilized but fail to complete meiosis. A small number of escaper embryos initiate development and display a range of phenotypes suggesting functions in both preblastoderm mitosis and head development. Our analysis of deadhead's RNA expression pattern is consistent with its maternal effect function: the RNA is predominately expressed in the nurse cells of the ovary, is maternally deposited into the egg, but does not appear to be zygotically expressed during embryogenesis. Thus both our genetic and molecular data are consistent with a function during meiosis and preblastoderm mitosis. Whether the head defect indicates an additional function or is an indirect consequence of earlier defects remains to be determined.

Sequence of the clathrin heavy chain from Saccharomyces cerevisiae and requirement of the COOH terminus for clathrin function.

The sequence of the clathrin heavy chain gene, CHC1, from Saccharomyces cerevisiae is reported. The gene encodes a protein of 1,653 amino acids that is 50% identical to the rat clathrin heavy chain (HC) (Kirchhausen, T., S. C. Harrison, E. P. Chow, R. J. Mattaliano, R. L. Ramachandran, J. Smart, and J. Brosius. 1987. Proc. Natl. Acad. Sci. USA. 84:8805-8809). The alignment extends over the complete length of the two proteins, except for a COOH-terminal extension of the rat HC and a few small gaps, primarily in the globular terminal domain. The yeast HC has four prolines in the region of the rat polypeptide that was proposed to form the binding site for clathrin light chains via an alpha-helical coiled-coil interaction. The yeast protein also lacks the COOH-terminal Pro-Gly rich segment present in the last 45 residues of the rat HC, which were proposed to be involved in the noncovalent association of HCs to form trimers at the triskelion vertex. To examine the importance of the COOH terminus of the HC for clathrin function, a HC containing a COOH-terminal deletion of 57 amino acids (HC delta 57) was expressed in clathrin-deficient yeast (chc1-delta). HC delta 57 rescued some of the phenotypes (slow growth at 30 degrees, genetic instability, and defects in mating and sporulation) associated with the chc1-delta mutation to normal or near normal. Also, truncated HCs were assembled into triskelions. However, cells with HC delta 57 were temperature sensitive for growth and still displayed a major defect in processing of the mating pheromone alpha-factor. Fewer coated vesicles could be isolated from cells with HC delta 57 than cells with the wild-type HC. This suggests that the COOH-terminal region is not required for formation of trimers, but it may be important for normal clathrin-coated vesicle structure and function.