Vitellogenin expression corresponds with reproductive status and caste in a primitively eusocial bee.

Vitellogenin (vg) expression is consistently associated with variation in insect phenotypes, particularly egg-laying. Primitively eusocial species, such as eusocial sweat bees, have behaviourally totipotent castes, in which each female is capable of high levels of ovarian development. Few studies have investigated vg expression patterns in primitively eusocial insects, and only one study has focused on a primitively eusocial bee. Here we use a primitively eusocial sweat bee, Lasioglossum laevissimum, and Real Time quantitative PCR (RT-qPCR) to investigate the relationship between vg expression, castes, and variation in phenotypes associated with castes differences. These assays showed that females with high ovarian development had the highest levels of vg expression, and that vg expression levels reflected the reproductive status of females first and caste second. This is in contrast to vg expression patterns observed in advanced eusocial queens and workers, which differ in vg expression based on caste and have caste-specific vg expression patterns. Furthermore, future queens (gynes) do not have ovarian development and had similar vg expression levels to early spring foundresses, which do have ovarian development, supporting Vg's function as a transporter of lipids and amino acids before diapause.

The adaptive significance of unproductive alternative splicing in primates.

Alternative gene splicing is pervasive in metazoa, particularly in humans, where the majority of genes generate splice variant transcripts. Characterizing the biological significance of alternative transcripts is methodologically difficult since it is impractical to assess thousands of splice variants as to whether they actually encode proteins, whether these proteins are functional, or whether transcripts have a function independent of protein synthesis. Consequently, to elucidate the functional significance of splice variants and to investigate mechanisms underlying the fidelity of mRNA splicing, we used an indirect approach based on analyzing the evolutionary conservation of splice variants among species. Using DNA polymerase ß as an indicator locus, we cloned and characterized the types and frequencies of transcripts generated in primary cell lines of five primate species. Overall, we found that in addition to the canonical DNA polymerase ß transcript, there were 25 alternative transcripts generated, most containing premature terminating codons. We used a statistical method borrowed from community ecology to show that there is significant diversity and little conservation in alternative splicing patterns among species, despite high sequence similarity in the underlying genomic (exonic) sequences. However, the frequency of alternative splicing at this locus correlates well with life history parameters such as the maximal longevity of each species, indicating that the alternative splicing of unproductive splice variants may have adaptive significance, even if the specific RNA transcripts themselves have no function. These results demonstrate the validity of the phylogenetic conservation approach in elucidating the biological significance of alternative splicing.

Evidence of the modulation of mRNA splicing fidelity in humans by oxidative stress and p53.

The majority of human genes generate mRNA splice variants and while there is little doubt that alternative splicing is an important biological phenomenon, it has also become apparent that some splice variants are associated with disease. To elucidate the molecular mechanisms responsible for generating aberrant splice variants, we have investigated alternative splicing of the human genes HPRT and POLB following oxidative stress in different genetic backgrounds. Our study revealed that splicing fidelity is sensitive to oxidative stress. Following treatment of cells with H2O2, the overall frequency of aberrant, unproductive splice variants increased in both loci. At least in POLB, splicing fidelity is p53 dependent. In the absence of p53, the frequency of POLB splice variants is elevated but oxidative stress does not further increase the frequency of splice variants. Our data indicate that mis-splicing following oxidative stress represents a novel and significant genotoxic outcome and that it is not simply DNA lesions induced by oxidative stress that lead to mis-splicing but changes in the alternative splicing machinery itself.

A survey of splice variants of the human hypoxanthine phosphoribosyl transferase and DNA polymerase beta genes: products of alternative or aberrant splicing?

Errors during the pre-mRNA splicing of metazoan genes can degrade the transmission of genetic information, and have been associated with a variety of human diseases. In order to characterize the mutagenic and pathogenic potential of mis-splicing, we have surveyed and quantified the aberrant splice variants in the human hypoxanthine phosphoribosyl transferase (HPRT) and DNA polymerase beta (POLB) in the presence and the absence of the Nonsense Mediated Decay (NMD) pathway, which removes transcripts with premature termination codons. POLB exhibits a high frequency of splice variants (40-60%), whereas the frequency of HPRT splice variants is considerably lower (approximately 1%). Treatment of cells with emetine to inactivate NMD alters both the spectrum and frequency of splice variants of POLB and HPRT. It is not certain at this point, whether POLB and HPRT splice variants are the result of regulated alternative splicing processes or the result of aberrant splicing, but it appears likely that at least some of the variants are the result of splicing errors. Several mechanisms that may contribute to aberrant splicing are discussed.