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Becker, Jeffrey M.

Publications and source records attributed to Becker, Jeffrey M..

UV Laser-Induced, Time-Resolved Transcriptome Responses of Saccharomyces cerevisiae

We observed the effect on gene transcription of laser-mediated, long-wavelength UV-irradiation of Saccharomyces cerevisiae by RNAseq analysis at times T15, T30, and T60 minutes after recovery in growth medium. Laser-irradiated cells were viable, and the transcriptional response was transient, with over 400 genes differentially expressed at T15 or T30, returning to basal level transcription by T60. Identification of transcripts exhibiting enhanced differential expression that were unique to UV laser-irradiation were identified by imposing a stringent significance cut-off (p <0.05, log 2 difference >2) then filtering out genes known as environmental stress response (ESR) genes. Using these rigorous criteria, 56 genes were differentially expressed at T15; at T30 differential expression was observed for 57 genes, some of which persisted from T15. Among the highly up-regulated genes were those supporting amino acid metabolic processes sulfur amino acids, methionine, aspartate, cysteine, serine), sulfur regulation (hydrogen sulfite metabolic processes, sulfate assimilation, sulfate reduction), proteasome components, amino acid transporters, and the iron regulon. At T30, the expression profile shifted to expression of transcripts related to catabolic processes (oxidoreductase activity, peptidase activity). Transcripts common to both T15 and T30 suggested an up-regulation of catabolic events, including UV damage response genes, and protein catabolism via proteasome and peptidase activity. Specific genes encoding tRNAs were among the down-regulated genes adding to the suggestion that control of protein biosynthesis was a major response to long-wave UV laser irradiation. These transcriptional responses bring light to the remarkable ability of the yeast cell to respond to a UV-induced environmental insult.

59 BASIC BIOLOGICAL SCIENCES↗

Identification of peptide-binding sites within BSA using rapid, laser-induced covalent cross-linking combined with high-performance mass spectrometry

In this paper, we are developing a rapid, time-resolved method using laser-activated cross-linking to capture protein-peptide interactions as a means to interrogate the interaction of serum proteins as delivery systems for peptides and other molecules. A model system was established to investigate the interactions between bovine serum albumin (BSA) and 2 peptides, the tridecapeptide budding-yeast mating pheromone (α-factor) and the decapeptide human gonadotropin-releasing hormone (GnRH). Cross-linking of α-factor, using a biotinylated, photoactivatable p-benzoyl-L-phenylalanine (Bpa)–modified analog, was energy-dependent and achieved within seconds of laser irradiation. Protein blotting with an avidin probe was used to detect biotinylated species in the BSA-peptide complex. The cross-linked complex was trypsinized and then interrogated with nano-LC–MS/MS to identify the peptide cross-links. Cross-linking was greatly facilitated by Bpa in the peptide, but some cross-linking occurred at higher laser powers and high concentrations of a non-Bpa–modified α-factor. This was supported by experiments using GnRH, a peptide with sequence homology to α-factor, which was likewise found to be cross-linked to BSA by laser irradiation. Analysis of peptides in the mass spectra showed that the binding site for both α-factor and GnRH was in the BSA pocket defined previously as the site for fatty acid binding. This model system validates the use of laser-activation to facilitate cross-linking of Bpa-containing molecules to proteins. Finally, the rapid cross-linking procedure and high performance of MS/MS to identify cross-links provides a method to interrogate protein-peptide interactions in a living cell in a time-resolved manner.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

An efficient temporal logic for robotic task planning

Computations required for temporal reasoning can be prohibitively expensive if fully general representations are used. Overly simple representations, such as totally ordered sequence of time points, are inadequate for use in a nonlinear task planning system. A middle ground is identified which is general enough to support a capable nonlinear task planner, but specialized enough that the system can support online task planning in real time. A Temporal Logic System (TLS) was developed during the Intelligent Task Automation (ITA) project to support robotic task planning. TLS is also used within the ITA system to support plan execution, monitoring, and exception handling.

Becker, Jeffrey M.↗