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The ability to express genes ectopically in bacteria is essential for diverse academic and industrial applications. Two major considerations when utilizing regulated promoter systems for ectopic gene expression are (1) the ability to titrate gene expression by addition of an exogenous inducer and (2) the leakiness of the promoter element in the absence of the inducer. Here, we describe a modular chromosomally integrated platform for ectopic gene expression in Vibrio cholerae. We compare the broadly used promoter elements P tac and P BAD to versions that have an additional theophylline-responsive riboswitch (P tac -riboswitch and P BAD -riboswitch). These constructs all exhibited unimodal titratable induction of gene expression, however, max induction varied with P tac  > P BAD  > P BAD -riboswitch > P tac -riboswitch. We also developed a sensitive reporter system to quantify promoter leakiness and show that leakiness for P tac  > P tac -riboswitch > P BAD ; while the newly developed P BAD -riboswitch exhibited no detectable leakiness. We demonstrate the utility of the tightly inducible P BAD -riboswitch construct using the dynamic activity of type IV competence pili in V. cholerae as a model system. The modular chromosomally integrated toolkit for ectopic gene expression described here should be valuable for the genetic study of V. cholerae and could be adapted for use in other species.

Somatic embryogenesis receptor kinases (SERKs) belong to a small gene family of receptor-like kinases involved in signal transduction. A total of 54 genes were shortlisted from the wheat genome survey sequence of which 5 were classified as SERKs and 49 were identified as SERK-like (SERLs). Tissue- specific expression of TaSERKs at major developmental stages of wheat corroborates their indispensable role during somatic and zygotic embryogenesis. TaSERK transcripts show inherent differences in their hormonal sensitivities, i.e. TaSERK2 and TaSERK3 elicits auxin- specific responses while TaSERK1, 4 and 5 were more specific towards BR-mediated regulation. The ectopic expression of TaSERK1, 2, 3, 4 and 5 in Arabidopsis led to enhanced plant height, larger silique size and increased seed yield. Zygotic embryogenesis specific genes showed a differential pattern in TaSERK Arabidopsis transgenics specifically in the silique tissues. Elongated hypocotyls and enhanced root growth were observed in the overexpression transgenic lines of all five TaSERKs. The inhibitory action of auxin and brassinosteroid in all the TaSERK transgenic lines indicates their role in regulating root development. The results obtained imply redundant functions of TaSERKs in maintaining plant growth and development.

Key flowering genes, FD and FD PARALOGUE (FDP) encoding bZIP transcription factors that interact with a FLOWERING LOCUS T (FT) in Arabidopsis were ectopically expressed in rice since we found AtFD and AtFDP also interact with HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1). Transgenic rice plants overexpressing AtFD and AtFDP caused reduction in plant height and spikelet size with decreased expression of genes involved in cell elongation without significant flowering time alteration in spite of increased expression of OsMADS14 and OsMADS15, rice homologues of APETALA1 (AP1) in the leaves. Simultaneous overexpression of AtFD and AtFDP enhanced phenotypes seen with overexpression of either single gene while transgenic rice plants expressing AtFD or AtFDP under the control of phloem-specific Hd3a promoter were indistinguishable from wild-type rice. Candidate genes responsible for the phenotypes were identified by comparison of microarray hybridization and their expression pattern was also examined in WT and transgenic rice plants. It has so far not been reported that AtFD and AtFDP affect cell elongation in plants, and our findings provide novel insight into the possible roles of AtFD and AtFDP in the mesophyll cells of plants, and potential genetic tools for manipulation of crop architecture.

Dedifferentiated liposarcoma (DDLPS) is a highly malignant subtype of human liposarcoma (LPS), whose genomic profile is characterized by chromosomal amplification at 12q13-q22. miR-26a-2 is one of the most frequently amplified genes in the region, and inhibition of its downstream target genes likely contributes to LPS tumorigenesis. Our previous study of LPS predicted homeobox protein A5 (HOXA5) as a target of miR-26a-2, and here we explored further the function of HOXA5, and its relationship with miR-26a-2 in DDLPS cells. Compared to normal human adipocytes, all LPS cell lines showed significant downregulation of HOXA5 (p = 0.046), and inhibition of miR-26a-2 using anti-miR-26a-2 substantially upregulated HOXA5 expression in these LPS cells. Interestingly, overexpression of HOXA5 alone induced very strong apoptotic response of LPS cells. HOXA5-induced apoptosis was p53-independent and caspase-dependent. Surprisingly, overexpression of HOXA5 induced nuclear translocation of RELA (p65), which was not associated with the transcriptional activity of RELA. Rather, nucleolar sequestration of RELA was observed. Overall, our study demonstrated for the first time that the downregulation of HOXA5 in LPS cells, partly by overexpression of miR-26a-2 in DDLPS, confers LPS cells resistance to apoptotic death. Further studies are required to understand the relationship of HOXA5 and the NFκB pathway in LPS cells.