An integrated computational pipeline and database to support whole-genome sequence annotation.

We describe here our experience in annotating the Drosophila melanogaster genome sequence, in the course of which we developed several new open-source software tools and a database schema to support large-scale genome annotation. We have developed these into an integrated and reusable software system for whole-genome annotation. The key contributions to overall annotation quality are the marshalling of high-quality sequences for alignments and the design of a system with an adaptable and expandable flexible architecture.

A Drosophila complementary DNA resource.

Collections of nonredundant, full-length complementary DNA (cDNA) clones for each of the model organisms and humans will be important resources for studies of gene structure and function. We describe a general strategy for producing such collections and its implementation, which so far has generated a set of cDNAs corresponding to over 40% of the genes in the fruit fly Drosophila melanogaster.

The Drosophila Numb protein inhibits signaling of the Notch receptor during cell-cell interaction in sensory organ lineage.

Specification of unequal daughter cell fates in the Drosophila external sense organ lineage requires asymmetric localization of the intrinsic determinant Numb as well as cell-cell interactions mediated by the Delta ligand and Notch receptor. Previous genetic studies indicated that numb acts upstream of Notch, and biochemical studies revealed that Numb can bind Notch. For a functional assay of the action of Numb on Notch signaling, we expressed these proteins in cultured Drosophila cells and used nuclear translocation of Suppressor of Hairless [Su(H)] as a reporter for Notch activity. We found that Numb interfered with the ability of Notch to cause nuclear translocation of Su(H); both the C-terminal half of the phosphotyrosine binding domain and the C terminus of Numb are required to inhibit Notch. Overexpression of Numb during wing development, which is sensitive to Notch dosage, revealed that Numb is also able to inhibit the Notch receptor in vivo. In the external sense organ lineage, the phosphotyrosine binding domain of Numb was found to be essential for the function but not for asymmetric localization of Numb. Our results suggest that Numb determines daughter cell fates in the external sense organ lineage by inhibiting Notch signaling.

Chimeric transcriptional activators generated in vivo from VnfA and AnfA of Azotobacter vinelandii: N-terminal domain of AnfA is responsible for dependence on nitrogenase Fe protein.

In vivo recombinants generating chimeras between the transcriptional activators VnfA and AnfA of Azotobacter vinelandii were constructed by cloning their structural genes in tandem and selecting against a conditionally lethal gene inserted between them. The parent molecules differ in their promoter specificities and in that AnfA, but not VnfA, requires the Fe protein of nitrogenase for its activity. Chimeras with fusion junctions in the N-terminal half of the central domain were found to be inactive, probably as a result of misfolding. All chimeras carrying the C-terminal domain of AnfA showed the corresponding promoter specificity, supporting the model which ascribes promoter specificity to the DNA-binding properties of the C-terminal domain. None of the chimeras showed the dependence on Fe protein typical of AnfA, including one which composed 82% of AnfA with only a short segment of VnfA at the N terminus. Deleting the N-terminal domain of AnfA gave a fully active protein which was also independent of Fe protein. This indicates that the N-terminal domain has an inhibitory effect on activity which is relieved by Fe protein.

Impact of stress and triiodothyronine on plasma magnesium fractions.

The differential regulation of free and bound plasma magnesium was studied in healthy volunteers exposed to various forms of stress, and patients screened for thyroid disorder. Both ergometric (7 min) and psychological (45 min) stress had no effect on free plasma magnesium, but increased the bound fraction. Before combined physical and psychological stress (45 min aerobatics) there was no correlation between the plasma fractions of magnesium; thereafter, there was a strong negative correlation between increased free and decreased bound magnesium. Three days of training in ground combat increased significantly the plasma levels of both magnesium fractions. Patients screened for thyroid disorder had a significant, positive correlation between both plasma fractions of magnesium, and a highly significant negative correlation between plasma T3 and the two magnesium fractions. No clear correlation between plasma catecholamines and magnesium levels was seen under any of the above conditions. Overall, the data show that the evaluation of the functions of circulating magnesium requires the specific measurement of the free and bound fractions.