Event-specific qualitative polymerase chain reaction analysis for two T-DNA copies in genetically modified orange Petunia

In 2017, various orange coloured petunia on the market turned out to be genetically modified (GM) without an official authorization for commercialization. Sequence analysis suggested these undeclared plants most probably originated from a plant transformation experiment performed in the 1980s. For a deeper understanding how GM petunia entered classical breeding programmes worldwide, and whether they originated from a single source or not, we undertook a molecular genetic characterization of the T-DNA integration sites in different GM petunia cultivars and breeding lines. By means of genome walking, we isolated different T-DNA sequences, which are located at the junctions between the T-DNA(s) and the petunia DNA. Based on the results obtained we conclude that there are at least two T-DNA copies of different lengths. This is supported by Southern blot analysis. For T-DNA1, the 3′-junction sequence was isolated, whereas the 5′-junction remained unclear. In contrast, for T-DNA2, the 5′-junction sequence was isolated, whereas the sequence isolated from the 3′-region consists only of T-DNA, but did not include the junction from the T-DNA to the petunia DNA. We developed primers for event-specific PCRs and screened a set of three orange GM petunia cultivars and 126 GM offspring from a commercial breeding program. We show that both T-DNA copies are present in all our tested GM petunia samples, which underpins the assumption of a single transgenic origin of the undeclared GM petunia. Most likely, the two T-DNAs are integrated in close proximity into the petunia genome.

An Isoform of the Oncogenic Splice Variant AIMP2-DX2 Detected by a Novel Monoclonal Antibody

AIMP2-DX2, an exon 2-deleted splice variant of AIMP2 (aminoacyl-tRNA synthetase-interacting multifunctional protein 2), is highly expressed in lung cancer and involved in tumor progression in vivo. Oncogenic function of AIMP2-DX2 and its correlation with poor prognosis of cancer patients have been well established; however, the application of this potentially important biomarker to cancer research and diagnosis has been hampered by a lack of antibodies specific for the splice variant, possibly due to the poor immunogenicity and/or stability of AIMP2-DX2. In this study a monoclonal antibody, H5, that specifically recognizes AIMP2-DX2 and its isoforms was generated via rabbit immunization and phage display techniques, using a short peptide corresponding to the exon 1/3 junction sequence as an antigen. Furthermore, based on mutagenesis, limited cleavage, and mass spectrometry studies, it is also suggested that the endogenous isoform of AIMP2-DX2 recognized by H5 is produced by proteolytic cleavage of 33 amino acids from N-terminus and is capable of inducing cell proliferation similarly to the uncleaved protein. H5 monoclonal antibody is applicable to enzyme-linked immunosorbent assay, immunoblot, immunofluorescence, and immunohistochemistry, and expected to be a valuable tool for detecting AIMP2-DX2 with high sensitivity and specificity for research and diagnostic purposes.

Rolling Circle cDNA Synthesis Uncovers Circular RNA Splice Variants

High-throughput RNA sequencing and novel bioinformatic pipelines have identified thousands of circular (circ)RNAs containing backsplice junction sequences. However, circRNAs generated from multiple exons may contain different combinations of exons and/or introns arising from alternative splicing, while the backsplice junction sequence is the same. To be able to identify circRNA splice variants, we developed a method termed circRNA-Rolling Circle Amplification (circRNA-RCA). This method detects full-length circRNA sequences by performing reverse transcription (RT) in the absence of RNase H activity, followed by polymerase chain reaction (PCR) amplification of full-length circRNAs using a forward primer spanning the backsplice junction sequence and a reverse primer exactly upstream of the forward primer. By sequencing the PCR products, circRNA splice variants bearing the same backsplice junctions, which were otherwise only predicted computationally, could be experimentally validated. The splice variants were further predicted to associate with different subsets of target RNA-binding proteins and microRNAs, supporting the notion that different circRNA splice variants can have different biological impacts. In sum, the circRNA-RCA method allows the accurate identification of full-length circRNA sequences, offering unique insight into their individual function.

Diversity of thefsr-gelERegion of theEnterococcus faecalisGenome but Conservation in Strains with Partial Deletions of thefsrOperon

ABSTRACTMostEnterococcus faecalisisolates carrygelE, but many are gelatinase nonproducers due to the lack offsrC(EF_1820) to EF_1841 (fsrC-EF_1841; 23.9 kb in strain V583), including most of the locus encoding Fsr, which activatesgelEexpression. Analysis of 22 accessibleE. faecalisgenomes revealed the identity of the 53-amino-acid propeptide offsrDacross multiple MLSTs (multilocus sequence types), although 12 distinctly different variations were found in the EF_1814-to-EF_1902 region. Diversity was seen infsrABC, in the region EF_1814 to EF_1902, and in a 700-kb region surroundingfsrC-EF_1841. However, analysis of five sequenced strains carrying thefsrC-EF_1841 deletion and the putative integrative conjugative element efaB5 showed almost identical single nucleotide polymorphisms (SNPs) ingelEand an identical junction sequence, despite their unrelated MLSTs, in contrast to those shown by strains without the deletion. Further analysis confirmed the conservedgelESNPs in 6 additional strains (11 in total) with the deletion. While we were unable to detect evidence of spontaneous deletion using OG1RF and 8 other strains, we were able to engineer a deletion of the 37-kbfsrC-EF_1841 region of OG1RF without deleterious effects, and the 37-kb mutant showed changes in biofilm and chaining similar to those shown byfsr-gelEmutants. In conclusion, we describe the identity offsrDdespite high plasticity within thefsrC-EF_1841 region and the surrounding sequence. However, strains lacking thefsrC-EF_1841 region show a distinct conservation of the sequence surrounding this deletion and ingelE, suggesting that the deletion may result from horizontal transfer and recombination.