Visual Cues Given by Humans Are Not Sufficient for Asian Elephants (Elephas maximus) to Find Hidden Food

Recent research suggests that domesticated species – due to artificial selection by humans for specific, preferred behavioral traits – are better than wild animals at responding to visual cues given by humans about the location of hidden food. \Although this seems to be supported by studies on a range of domesticated (including dogs, goats and horses) and wild (including wolves and chimpanzees) animals, there is also evidence that exposure to humans positively influences the ability of both wild and domesticated animals to follow these same cues. Here, we test the performance of Asian elephants (Elephas maximus) on an object choice task that provides them with visual-only cues given by humans about the location of hidden food. Captive elephants are interesting candidates for investigating how both domestication and human exposure may impact cue-following as they represent a non-domesticated species with almost constant human interaction. As a group, the elephants (n = 7) in our study were unable to follow pointing, body orientation or a combination of both as honest signals of food location. They were, however, able to follow vocal commands with which they were already familiar in a novel context, suggesting the elephants are able to follow cues if they are sufficiently salient. Although the elephants’ inability to follow the visual cues provides partial support for the domestication hypothesis, an alternative explanation is that elephants may rely more heavily on other sensory modalities, specifically olfaction and audition. Further research will be needed to rule out this alternative explanation.     

Substitution by Inosine at the 3��-Ultimate and Penultimate Positions of 16S rRNA Gene Universal Primers

Universal 16S rRNA gene primers (8F and 518R) bearing inosine substitutions at either the 3??-ultimate or the 3??-ultimate and penultimate base positions were exploited for the first time to study the bacterial community associated with coral polymicrobial Black Band Disease (BBD). Inosine-modified universal primer pairs display some shifting in the composition of 16S rRNA gene libraries, as well as expanding the observed diversity of a BBD bacterial community at the family/class level. Possible explanations for the observed shifts are discussed. These results thus point to the need for adopting multiple approaches in designing 16S rRNA universal primers for PCR amplification and subsequent construction of 16S rRNA gene libraries or pyrosequencing in the exploration of complex microbial communities.     

Identification of adult mineralized tissue zebrafish mutants

Zebrafish craniofacial, skeletal, and tooth development closely resembles that of higher vertebrates. Our goal is to identify viable adult zebrafish mutants that can be used as models for human mineralized craniofacial, dental, and skeletal system disorders. We used a large-scale forward-genetic chemical N-ethyl-nitroso-urea mutagenesis screen to identify 17 early lethal homozygous recessive mutants with defects in craniofacial cartilage elements, and 7 adult homozygous recessive mutants with mineralized tissue phenotypes including craniofacial shape defects, fused sutures, dysmorphic or missing skeletal elements, scoliosis, and neural arch defects. One mutant displayed both an early lethal homozygous phenotype and an adult heterozygous phenotype. These results extend the utility of the zebrafish model beyond the embryo to study human bone and cartilage disorders.     

Formulating a fluorogenic assay to evaluate S-adenosyl-L-methionine analogues as protein methyltransferase cofactors

Protein methyltransferases (PMTs) catalyze arginine and lysine methylation of diverse histone and nonhistone targets. These posttranslational modifications play essential roles in regulating multiple cellular events in an epigenetic manner. In the recent process of defining PMT targets, S-adenosyl-L-methionine (SAM) analogues have emerged as powerful small molecule probes to label and profile PMT targets. To examine efficiently the reactivity of PMTs and their variants on SAM analogues, we transformed a fluorogenic PMT assay into a ready high throughput screening (HTS) format. The reformulated fluorogenic assay is featured by its uncoupled but more robust character with the first step of accumulation of the commonly-shared reaction byproduct S-adenosyl-L-homocysteine (SAH), followed by SAH-hydrolase-mediated fluorogenic quantification. The HTS readiness and robustness of the assay were demonstrated by its excellent Z' values of 0.83-0.95 for the so-far-examined 8 human PMTs with SAM as a cofactor (PRMT1, PRMT3, CARM1, SUV39H2, SET7/9, SET8, G9a and GLP1). The fluorogenic assay was further implemented to screen the PMTs against five SAM analogues (allyl-SAM, propargyl-SAM, (E)-pent-2-en-4-ynyl-SAM (EnYn-SAM), (E)-hex-2-en-5-ynyl-SAM (Hey-SAM) and 4-propargyloxy-but-2-enyl-SAM (Pob-SAM)). Among the examined 8 × 5 pairs of PMTs and SAM analogues, native SUV39H2, G9a and GLP1 showed promiscuous activity on allyl-SAM. In contrast, the bulky SAM analogues, such as EnYn-SAM, Hey-SAM and Pob-SAM, are inert toward the panel of human PMTs. These findings therefore provide the useful structure-activity guidance to further evolve PMTs and SAM analogues for substrate labeling. The current assay format is ready to screen methyltransferase variants on structurally-diverse SAM analogues.     

Characterization of the gene for fructose-1,6-bisphosphatase from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Sequence, protein homology, and expression during growth on glucose

We have determined the nucleotide sequence of the gene for fructose-1,6-bisphosphatase from both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The predicted protein sequence for fructose-1,6-bisphosphatase from S. cerevisiae contains 347 amino acids and has a molecular weight of 38,100; that from S. pombe, contains 346 amino acids and has a molecular weight of 38,380. Comparison of these amino acid sequences with each other and that of pig kidney fructose-1,6-bisphosphatase shows several regions of strong homology separated by regions of divergence. These homologous regions are likely candidates for functional domains. A gene cassette was constructed for fructose-1,6-bisphosphatase from S. cerevisiae and the gene cassette expressed from the regulated PHO5 and GAL1 promoters of yeast. Yeast cells expressing fructose-1,6-bisphosphatase, while growing on glucose, accumulated large amounts of enzyme intracellularly, suggesting that glucose-regulated proteolytic inactivation does not operate efficiently under these conditions. Growth on glucose was not inhibited by the expression of fructose 1,6-bisphosphatase.