No abstract

The link between polyadenylation (pA) and various biological, behavioral, and pathological events of eukaryotes underlines the need to develop in vivo polyadenylation assay methods for characterization of the cis-acting elements, trans-acting factors and environmental stimuli that affect polyadenylation efficiency and/or relative usage of two alternative polyadenylation (APA) sites. The current protein-based CAT or luciferase reporter systems can measure the polyadenylation efficiency of a single pA site or candidate cis element but not the choice of two APA sites. To address this issue, we developed a set of four new bicistronic reporter vectors that harbor either two luciferase or fluorescence protein open reading frames connected with one Internal Ribosome Entry Site (IRES). Transfection of single or dual insertion constructs of these vectors into mammalian cells demonstrated that they could be utilized not only to quantify the strength of a single candidate pA site or cis element, but also to accurately measure the relative usage of two APA sites at both the mRNA (qRT-PCR) and protein levels. This represents the first reporter system that can study polyadenylation efficiency of a single pA site or element and regulation of two APA sites at both the mRNA and protein levels.

Helicoverpa armigera is a globally-important crop pest with a WZ (female)/ZZ (male) sex chromosome system. The absence of discernible sexual dimorphism in its egg and larval stages makes it impossible to address any sex-related theoretical and applied questions before pupation unless a W-specific sequence marker is available for sex diagnosis. To this end, we used one pair of morphologically pre-sexed pupae to PCR-screen 17 non-transposon transcripts selected from 4855 W-linked candidate reads identified by mapping a publicly available egg transcriptome of both sexes to the male genome of this species and detected the read SRR1015458.67499 only in the female pupa. Subsequent PCR screenings of this read and the previously reported female-specific RAPD (random amplified polymorphic DNA) marker AF18 with ten more pairs of pre-sexed pupae and different annealing positions and/or temperatures as well as its co-occurrence with the female-specific transcript splicing isoforms of doublesex gene of H. armigera (Hadsx) and amplification and sequencing of their 5 unknown flanking sequences in three additional pairs of pre-sexed pupae verified that SRR1015458.67499 is a single copy protein-coding gene unique to W chromosome (named GUW1) while AF18 is a multicopy MITE transposon located on various chromosomes. Test application of GUW1 as a marker to sex 30 neonates of H. armigera yielded a female/male ratio of 1.14: 1.00. Both GUW1 and Hadsx splicing isoforms assays revealed that the H. armigera embryo cell line QB-Ha-E-1 is a male cell line. Taken together, GUW1 is not only a reliable DNA marker for sexing all stages of H. armigera and its cell lines, but also represents the first W-specific protein-coding gene in lepidopterans.