Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization

The effects of photocaged nucleosides on the DNA polymerization reaction was investigated, finding that most polymerases are unable to recognize and read through the presence of a single caging group on the DNA template. Based on this discovery, a new method of introducing mutations into plasmid DNA via a light-mediated mutagenesis protocol was developed. This methodology is advantageous over several common approaches in that it requires the use of only two polymerase chain reaction primers, and does not require any restriction sites or use of restriction enzymes. Additionally, this approach enables not only site-directed mutations, but also the insertion of DNA strands of any length into plasmids and the deletion of entire genes from plasmids.     

Hemolymph amino acid variations following behavioral and genetic changes in individual Drosophila larvae

This study investigated the effect of different sampling environments on hemolymph amino acid content of individual Drosophila melanogaster larvae. Hemolymph was collected from individual third instar larvae under cold-anesthetized, awake, and stress conditions. Qualitative and quantitative hemolymph amino acid analyses were performed via capillary electrophoresis with laser-induced fluorescence detection. The hemolymph amino acid concentrations, particularly arginine, glutamate, and taurine, changed significantly depending on the prior-to-sample-collection environments. Hemolymph amino acid analyses of six different Drosophila genotypes including two control genotypes and four mutant alleles were also carried out. Two mutant genotypes with over and under expression of a putative cystine-glutamate exchanger subunit were significantly different from each other with respect to their hemolymph glutamate, glycine, lysine, and taurine levels. Hemolymph amino acid analyses of stressed larvae of two control and two mutant genotypes indicated that behavior-related hemolymph chemical changes are also genotype dependent.     

Biological aspects of Cuvier’s beaked whale (Ziphius cavirostris) recorded in the Croatian part of the Adriatic Sea

The paper describes two stranded ziphiids from Croatia: a subadult female (length 430 cm, body mass 610 kg) that was stranded in 2001 and an adult male (length 510 cm, body mass ∼1,000 kg) that was stranded in 2002. Both were confirmed to be Cuvier’s beaked whales (Ziphius cavirostris Cuvier, 1823) from analysis of mitochondrial DNA sequences and osteological features. There are no previous records of Cuvier’s beaked whales from the Croatian part of the Adriatic. The external shape of the head of the female specimen appears to be significantly different from the heads of Cuvier’s beaked whales from other seas. The Croatian specimen exhibited embedded pieces of gravel in the gum tissue around the tip of the lower and upper jaws, which was observed for the first time in a Cuvier’s beaked whale. The presence of the female in shallow coastal waters for several weeks and her boat-positive behaviour are apparently also first records of this kind for the species. The female was found to have ingested several plastic bags which likely caused her death. These are the northernmost findings of this species in the Adriatic Sea.     

Optochemical control of RNA interference in mammalian cells

Short interfering RNAs (siRNAs) and microRNAs (miRNAs) have been widely used in mammalian tissue culture and model organisms to selectively silence genes of interest. One limitation of this technology is the lack of precise external control over the gene-silencing event. The use of photocleavable protecting groups installed on nucleobases is a promising strategy to circumvent this limitation, providing high spatial and temporal control over siRNA or miRNA activation. Here, we have designed, synthesized and site-specifically incorporated new photocaged guanosine and uridine RNA phosphoramidites into short RNA duplexes. We demonstrated the applicability of these photocaged siRNAs in the light-regulation of the expression of an exogenous green fluorescent protein reporter gene and an endogenous target gene, the mitosis motor protein, Eg5. Two different approaches were investigated with the caged RNA molecules: the light-regulation of catalytic RNA cleavage by RISC and the light-regulation of seed region recognition. The ability to regulate both functions with light enables the application of this optochemical methodology to a wide range of small regulatory RNA molecules.     

Comprehensive Analysis of Varicella-Zoster Virus Proteins Using a New Monoclonal Antibody Collection

Varicella-zoster virus (VZV) is the etiological agent of chickenpox and shingles. Due to the virus''s restricted host and cell type tropism and the lack of tools for VZV proteomics, it is one of the least-characterized human herpesviruses. We generated 251 monoclonal antibodies (MAbs) against 59 of the 71 (83%) currently known unique VZV proteins to characterize VZV protein expression in vitro and in situ. Using this new set of MAbs, 44 viral proteins were detected by Western blotting (WB) and indirect immunofluorescence (IF); 13 were detected by WB only, and 2 were detected by IF only. A large proportion of viral proteins was analyzed for the first time in the context of virus infection. Our study revealed the subcellular localization of 46 proteins, 14 of which were analyzed in detail by confocal microscopy. Seven viral proteins were analyzed in time course experiments and showed a cascade-like temporal gene expression pattern similar to those of other herpesviruses. Furthermore, selected MAbs tested positive on human skin lesions by using immunohistochemistry, demonstrating the wide applicability of the MAb collection. Finally, a significant portion of the VZV-specific antibodies reacted with orthologs of simian varicella virus (SVV), thus enabling the systematic analysis of varicella in a nonhuman primate model system. In summary, this study provides insight into the potential function of numerous VZV proteins and novel tools to systematically study VZV and SVV pathogenesis.