Archive for May, 2009
Novel Splicing Generates Unique mRNA Isoforms from the Ribosomal Protein L22-like Gene in Drosophila melanogaster
Alternative splicing contributes to protein diversity by creating multiple mRNAs from a single gene that may encode functionally-distinct protein variants. Little is known about alternative splicing of ribosomal protein (rp) genes as a possible mechanism to generate ribosome diversity itself. As complex organelles, eukaryotic ribosomes contain several RNAs and ~80 rp components, each represented as a single copy. Paralogous proteins exist in several rp families and their presence may indicate functional redundancy or specialized functions. Incorporation of specialized paralogs into ribosomes could generate functionally-distinct classes of ribosomes, possibly dedicated to translation of specific mRNAs or to differential regulation of protein synthesis within cells/tissues/developmental stages. Drosophila rp L23a and L22 contain a unique N-terminal extension (of unknown function) with homology to histone H1 (Koyama et al., 1999). Gene expression profiling revealed a novel transcript called “L22-like” that displays an embryonic gonad-specific expression pattern by in situ hybridization compared to a constitutive pattern for L22 mRNA (Shigenobu et al., 2006; Kai et al., 2005). Noticeably, this paralog contains a similar histone H1- like domain, and if expressed at the protein level and assembled into ribosomes, could define a unique class of ribosomes within the fly gonad. Whether or not L22-like and L22 are interchangeable within the ribosome is unknown. Further insight into L22-like expression was determined by RT-PCR analysis using RNA from embryo, larval, and adult stages, as well as from S2 cells. Full-length L22-like mRNAs were detected at all stages. Surprisingly, lower molecular weight (MW) amplicons were also present. Cloning and sequencing revealed that the lower MW amplicons (designated “L22-like short”) are mRNA variants, in which non-canonical splice sites within an exon are used for RNA splicing to remove a previously unidentified intron. Northern blot analysis confirmed the presence of multiple L22-like mRNAs. Preliminary Western blot analyses show protein products of the predicted size for both L22-like and L22-like short isoforms in S2 cells. Whether or not the L22-like isoforms are incorporated into ribosomes is yet to be determined. Collectively, these experiments provide a foundation for understanding developmental regulation of L22-like rp gene expression and how rp variants may contribute to ribosome diversity within cells.
Retention and Release of Cryptosporidium parvum Oocysts by Experimental Biofilms Composed of Natural Stream Microbial Communities
Cryptosporidium is a group of waterborne protozoan parasites that cause significant gastrointestinal disease in humans. The potential exists for these parasites to accumulate in stream or pipe surface biofilms and to be subsequently released to contaminate the water supply. Natural microbial assemblages were collected from three streams for three seasons (fall, winter, and spring) and used to grow biofilms in laboratory microcosms. These biofilms were then exposed to Cryptosporidium parvum oocysts via continuous inflow to determine daily influx, efflux, and biofilm retention and sloughing. Daily oocyst counts showed that oocysts attached to the biofilm quickly (within hours) and released slowly (within days). In these experiments, at least 40% of the oocysts added to the system attached to the biofilm in the first 72 hours, and at least 20% of the oocysts remained attached to the biofilm beyond 72 hours after the oocyst supply was removed. Although variation across site and season exists, likely as a result of variable microbial communities, all biofilms captured at least 40% of the oocysts and retained some of those oocysts for at least five days. The biofilm attachment and detachment dynamics of C. parvum oocysts have important implications for public health and suggest that biofilm monitoring should be included in routine water supply monitoring.