Extended life-span and stress resistance in the Drosophila mutant methuselah.

Toward a genetic dissection of the processes involved in aging, a screen for gene mutations that extend life-span in Drosophila melanogaster was performed. The mutant line methuselah (mth) displayed approximately 35 percent increase in average life-span and enhanced resistance to various forms of stress, including starvation, high temperature, and dietary paraquat, a free-radical generator. The mth gene predicted a protein with homology to several guanosine triphosphate-binding protein-coupled seven-transmembrane domain receptors. Thus, the organism may use signal transduction pathways to modulate stress response and life-span.

Point mutations within and outside the homeodomain identify sequences required for proboscipedia homeotic function in Drosophila.

The Drosophila homeotic gene proboscipedia (pb) encodes a homeodomain protein homologous to vertebrate HoxA2/B2 required for adult mouthparts formation. A transgenic Hsp70-pb (HSPB) element that rescues pb mutations also induces the dominant transformation of antennae to maxillary palps. To identify sequences essential to PB protein function, we screened for EMS-induced HSPB mutations leading to phenotypic reversion of the HSPB transformation. Ten revertants harbor identified point mutations in HSPB coding sequences. The point mutations that remove all detectable phenotypes in vivo reside either within the homeodomain or, more unexpectedly, in evolutionarily nonconserved regions outside the homeodomain. Two independent homeodomain mutations that change the highly conserved Arginine-5 in the N-terminal hinge show effects on adult eye development, suggesting a previously unsuspected role for Arg5 in functional specificity. Three additional revertant mutations outside the homeodomain reduce but do not abolish PB+ activity, identifying protein elements that contribute quantitatively to pb function. This in vivo analysis shows that apart from the conserved motifs of PB, other elements throughout the protein make important contributions to homeotic function.

Ras1-mediated modulation of Drosophila homeotic function in cell and segment identity.

Mutations of the Drosophila homeotic proboscipedia gene (pb, the Hox-A2/B2 homologue) provoke dose-sensitive defects. These were used to search for dose-sensitive dominant modifiers of pb function. Two identified interacting genes were the proto-oncogene Ras1 and its functional antagonist Gap1, prominent intermediaries in known signal transduction pathways. Ras1+ is a positive modifier of pb activity both in normal and ectopic cell contexts, while the Ras1-antagonist Gap1 has an opposite effect. A general role for Ras1 in homeotic function is likely, since Ras1+ activity also modulates functions of the homeotic loci Sex combs reduced and Ultrabithorax. Our data suggest that the modulation occurs by a mechanism independent of transcriptional control of the homeotic loci themselves, or of the Ras1/Gap1 genes. Taken together our data support a role for Ras1-mediated cell signaling in the homeotic control of segmental differentiation.

A homeodomain point mutation of the Drosophila proboscipedia protein provokes eye loss independently of homeotic function.

The Drosophila homeotic gene proboscipedia (pb: HoxA2/B2 homolog) is required for adult mouthparts development. Ectopic PB protein expression from a transgenic Hsp70-pb minigene (HSPB) results in transformation of adult antennae to maxillary palps. In contrast, most tissues appear refractory to PB-induced effects. To study the basis of homeotic tissue specificity we are isolating and studying mutations that modify dominant HSPB-induced phenotypes. One HSPB point mutation (Arg5 of the homeodomain to His) removes homeotic activity in the mouthparts and antennae, but provokes a dose-sensitive eye loss. We show that eye loss can be induced by PB proteins that no longer effectively bind to DNA. The dose-sensitive eye loss thus appears to be mediated by specific, context-dependent protein-protein interactions.

Homeotic proboscipedia cell identity functions respond to cell signaling pathways along the proximo-distal axis.

To better understand how the different cell identities composing a segment are attributed and coordinated under the control of a single homeotic selector gene, we examined dose-sensitive homeotic phenotypes associated with gain-of-function and loss-of-function mutations of the homeotic gene proboscipedia (pb; Hox-A2/-B2). We then employed dose-sensitive segment and cell identity phenotypes resulting from ectopic proboscipedia expression to screen for other interacting loci. We find that pb, as well as the homeotic loci Ultrabithorax, Sex combs reduced and Antennapedia, respond to positional information along the proximo-distal axis. This response for pb implicates at least two signal transduction pathways, those involving Ras1 and Notch.

Genetic studies of cefotaxime resistance in Streptococcus pneumoniae: relationship to transformation deficiency.

A laboratory pneumococcal strain resistant to cefotaxime was studied by DNA-induced transformation in order to characterize its genetic structure. At least three independent genes were required to confer the highest level of resistance to this beta-lactam antibiotic. The accumulation of mutations in these three genes accounted for three levels of resistance. Mutation of the gene encoding penicillin-binding protein 2x was very likely responsible for the first step of resistance, which was a prerequisite for sequential increase in resistance. Additionally, strains highly resistant to cefotaxime were defective for natural transformation. Revertants of these strains were frequently observed. Such strains had recovered full transformability, suggesting a correlation between the inability to be transformed and a high level of resistance to cefotaxime. The possibility of electrotransforming these highly resistant strains suggests that natural transformation is probably blocked at the DNA-uptake level.

The role of penicillin-binding protein 3 (PBP 3) in cefotaxime resistance in Streptococcus pneumoniae.

A pneumococcal strain, with a reduced amount of penicillin-binding protein 3 (PBP 3), permitted an analysis of the role of this protein in cefotaxime resistance. We observed that reduced amounts of PBP 3 sensitize the bacteria to high temperature, to excess glycine and to some D-amino acids. These phenotypes suggest that the amount of PBP 3 may influence the membrane properties of the bacteria. The strain with reduced PBP 3 was transformed to cefotaxime resistance. We show that the PBP 3 mutation, in certain genetic backgrounds, decreases the level of resistance to cefotaxime by a factor of 2. Models are presented to explain this result.

One-ended insertion of IS911.

An apparently nonreplicative integration reaction mediated by the insertion sequence IS911 has been analyzed. It is shown to involve the right-end inverted repeat (IRR) of the element and sequences in the flanking vector DNA. The flanking sequences appear to behave as a surrogate IS911 end, since integration is greatly reduced when limited similarities with IRR are eliminated by site-directed mutagenesis. Data are presented which suggest that the activity of the IRR junction results from the proximity of the transposase gene and may therefore reflect preferential transposase recognition of IRR in cis.