Evaluation of Plant-Derived Promoters for Constitutive and Tissue-Specific Gene Expression in Potato.

Various plant-derived promoters can be used to regulate ectopic gene expression in potato. In the present study, four promoters derived from the potato genome have been characterized by the expression of identical cassettes carrying the fusion with the reporter ß-glucuronidase (gusA) gene. The strengths of StUbi, StGBSS, StPat, and StLhca3 promoters were compared with the conventional constitutive CaMV 35S promoter in various organs (leaves, stems, roots, and tubers) of greenhouse-grown plants. The final amount of gene product was determined at the post-transcriptional level using histochemical analysis, fluorometric measurements, and Western blot analysis. The promoter strength comparison demonstrated that the StUbi promoter generally provided a higher level of constitutive ß-glucuronidase accumulation than the viral CaMV 35S promoter. Although the StLhca3 promoter was predominantly expressed in a green tissue-specific manner (leaves and stems) while StGBSS and StPat mainly provided tuber-specific activity, a "promoter leakage" was also found. However, the degree of unspecific activity depended on the particular transgenic line and tissue. According to fluorometric data, the functional activity of promoters in leaves could be arranged as follows: StLhca3 > StUbi > CaMV 35S > StPat > StGBSS (from highest to lowest). In tubers, the higher expression was detected in transgenic plants expressing StPat-gusA fusion construct, and the strength order was as follows: StPat > StGBSS > StUbi > CaMV 35S > StLhca3. The observed differences between expression patterns are discussed considering the benefits and limitations for the usage of each promoter to regulate the expression of genes in a particular potato tissue.

Bicoid-Dependent Activation of the Target Gene hunchback Requires a Two-Motif Sequence Code in a Specific Basal Promoter.

In complex genetic loci, individual enhancers interact most often with specific basal promoters. Here we investigate the activation of the Bicoid target gene hunchback (hb), which contains two basal promoters (P1 and P2). Early in embryogenesis, P1 is silent, while P2 is strongly activated. In vivo deletion of P2 does not cause activation of P1, suggesting that P2 contains intrinsic sequence motifs required for activation. We show that a two-motif code (a Zelda binding site plus TATA) is required and sufficient for P2 activation. Zelda sites are present in the promoters of many embryonically expressed genes, but the combination of Zelda plus TATA does not seem to be a general code for early activation or Bicoid-specific activation per se. Because Zelda sites are also found in Bicoid-dependent enhancers, we propose that simultaneous binding to both enhancers and promoters independently synchronizes chromatin accessibility and facilitates correct enhancer-promoter interactions.

Engineered and Natural Promoters and Chromatin-Modifying Elements for Recombinant Protein Expression in CHO Cells.

Promoters play a pivotal role in integrating and processing the signals related to transcription initiation. Strong natural viral promoters, such as hCMV or SV40E, have been routinely employed to achieve a high rate of gene expression in ubiquitously used Chinese hamster ovary (CHO) cells. However, viral promoters are susceptible to epigenetic silencing and lack precise regulation levers. This has paved the way to more sensible control elements: endogenous, inducible, and synthetic promoters. In this review we summarize and discuss the use of natural viral, mammalian, and endogenous promoters, as well as recent advances in synthetic promoters and inducible systems for protein expression in CHO cells. Not only the level of transcription, but its long-term stability is crucial for recombinant protein production. Epigenetic chromatin-modifying elements, such as ubiquitously acting chromatin opening elements (UCOEs), matrix attachment regions (MARs), insulators and stabilizing anti-repressors (STARs) significantly improve transcription levels over extended cultivation time and are also discussed here. This review provides up-to date information to facilitate the choice of a suitable promoter and adjacent chromatin-modifying elements to maximize transgene expression as well as ensure long-term expression stability in CHO cell culture.

Comparison of the impact of viral and plant-derived promoters regulating selectable marker gene on maize transformation and transgene expression.

KEY MESSAGE: The choice of promoter regulating the selectable marker gene impacts transformation efficiency, copy number and the expression of selectable marker and flanking genes in maize. Viral or plant-derived constitutive promoters are often used to regulate selectable marker genes. We compared two viral promoters, cauliflower mosaic virus (CaMV 35T) and sugarcane bacilliform virus (SCBV) with two plant promoters, rice actin1 (OsAct1) and maize ubiquitin 1 (ZmUbi1) to drive aryloxyalkanoate dioxygenase (aad-1) selectable marker gene in maize inbred line B104. ZmUbi1- and OsAct1-containing constructs demonstrated higher transformation frequencies (43.8 and 41.4%, respectively) than the two viral promoter constructs, CaMV 35T (25%) and SCBV (8%). Interestingly, a higher percentage of single copy events were recovered for SCBV (82.1%) and CaMV 35T (59.3%) promoter constructs, compared to the two plant-derived promoters, OsAct1 (40.0%), and ZmUbi1 (27.6%). Analysis of protein expression suggested that the viral promoter CaMV 35T expressed significantly higher AAD-1 protein (174.6 ng/cm2) than the OsAct1 promoter (12.6 ng/cm2) in T0 leaf tissue. When measured in T2 callus tissue, the two viral promoters both had higher expression and more variability than the two plant-derived promoters. A potential explanation for why viral promoters produce lower transformation efficiencies but higher percentages of low copy number events is discussed. In addition, viral promoters regulating aad-1 were found to influence the expression of upstream flanking genes in both T0 leaf and T2 callus tissue.

Clustered protocadherins.

The majority of vertebrate protocadherin (Pcdh) genes are clustered in a single genomic locus, and this remarkable genomic organization is highly conserved from teleosts to humans. These clustered Pcdhs are differentially expressed in individual neurons, they engage in homophilic trans-interactions as multimers and they are required for diverse neurodevelopmental processes, including neurite self-avoidance. Here, we provide a concise overview of the molecular and cellular biology of clustered Pcdhs, highlighting how they generate single cell diversity in the vertebrate nervous system and how such diversity may be used in neural circuit assembly.