Olfactory Jump Reflex Habituation in Drosophila and Effects of Classical Conditioning Mutations

Habituation is a nonassociative learning mechanism, in which an initial response toward repeated stimuli gradually wanes. This is amongst the simplest and most widespread forms of behavioral plasticity. So far, neither the underlying molecular mechanisms nor the precise neural networks of habituation are well understood. We have developed a novel paradigm to quantify habituation of the olfactory jump reflex in Drosophila. We present data demonstrating several behavioral properties of this phenomenon, generally observed in other species. We also show that the dunce and rutabaga memory mutants behave abnormally in this assay, suggesting that this assay might be used in behavioral screens for new mutants with defects in this simpler form of behavioral plasticity

Transcriptional attenuation control of the tylosin-resistance gene tlrA in Streptomyces fradiae

The tylosin producer Streptomyces fradiae contains four known resistance genes, two of which (tlrA and tlrD) encode methyltransferases that act on ribosomal RNA at a common site. Expression of tlrA is regulated via transcriptional attenuation. A short transcript, only 411 nucleotides long, terminates 27 nucleotides into the methylase‐coding sequence in the uninduced state. Induction of tlrA is proposed to involve a ribosome‐mediated conformational change within the mRNA leader that allows transcription to continue beyond the attenuation site, resulting in a transcript about 1450 nucleotides long. Transplantation of tlrD and/or tlrA into Streptomyces albus revealed that the induction specificity of tlrA depends upon the state of the ribosomes and is significantly altered in strains also expressing tlrD

Enzyme Engineering for Nonaqueous Solvents: Random Mutagenesis to Enhance Activity of Subtilisin E in Polar Organic Media

Enzyme activity is often dramatically reduced in polar organic solvents, even under conditions where the folded structures are stable. We have utilized random mutagenesis by polymerase chain reaction (PCR) techniques combined with screening for enhanced activity in the presence of dimethylformamide (DMF) to probe mechanisms by which improved enzymes for chemical synthesis in polar organic media might be obtained. Two amino acid substitutions which enhance subtilisin E activity in the presence of DMF, Q103R and D60N, were identified by screening on agar plates containing DMF and casein. The two substitutions are located near the substrate binding pocket or in the active site, and their effects on the catalytic efficiency k_(cat)/K_M for the hydrolysis of a peptide substrate are additive. The effects of D60N are apparent only in the presence of DMF, highlighting the importance of screening in the organic solvent. Protein engineering is an effective approach to enhancing enzyme activity in organic media: the triple mutant D60N+Q103R+N218S is 38 times more active than wild–type subtilisin E in 85% DMF. An evolutionary approach consisting of multiple steps of random muta–genesis and screening in continually higher concentrations of organic solvent should result in enzymes that are substantially more active in organic media