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BBA - Gene Structure and Expression (v.1759, #6)

Editorial Board (pp. ii).

Cloning and functional analysis of the novel gene GhDBP3 encoding a DRE-binding transcription factor from Gossypium hirsutum by Bo Huang; Jin-Yuan Liu (pp. 263-269).

A novel cDNA encoding DRE-binding transcription factor, designated GhDBP3, was cloned from Gossypium hirsutum. This protein was classified into A-4 group of DREB subfamily based on multiple sequence alignment and phylogenetic characterization. Semiquantitative RT-PCR showed that GhDBP3 was expressed in the leaves, cotyledons, roots and stems of 2-week-old cotton seedlings under non-stress conditions and was greatly induced in the cotton cotyledons by drought, NaCl, low temperature and ABA treatment. EMSA revealed that GhDBP3 was able to bind to the DRE cis-element in vitro. Transient assay using the particle bombardment method showed that GhDBP3 was a transcriptional activator, capable of activating expression of a reporter gene driven by the LEA D113 promoter containing a DRE like sequence in tobacco cells. Our results indicate that GhDBP3 could be a new member of DRE-binding transcription factor family and may play an important role in response to ABA and environmental stresses.

Keywords: Abbreviations; DRE; dehydration responsive element; DBP; DRE-binding protein; ABA; Abscisic acid; CBF; C-repeat binding factor; EMSA; Electrophoretic Mobility Shift Assays; LEA; late-embryogenesis abundantCotton; DRE-binding protein; EMSA; Abiotic stress; Transcriptional activator

Genomic organization and expression of the human mono-ADP-ribosyltransferase ART3 gene by Maik Friedrich; Andreas Grahnert; Claudia Klein; Katrin Tschöp; Kurt Engeland; Sunna Hauschildt (pp. 270-280).

Here we describe an RT-PCR analysis of mono-ADP-ribosyltransferase 3 (ART3) mRNA expression in macrophages, testis, semen, tonsil, heart and skeletal muscle and the complete gene structure as obtained by sequence alignment of PCR products with a human genomic clone (GenBank accession no.AC112719). Twelve exons (ex1-12) were found to make up the coding region of the gene (one more than previously published). Two prominent classes of ART3 splice variants could be distinguished by the presence or absence of ex2 which encodes most of ART3 protein. Among the ex2-containing mRNA species, the most frequently amplified variant did not include exons 9 to 11, except in skeletal muscle, in which the major splice variant lacked ex10 only. Two different, previously not reported 5′ non-translated regions (5′ UTRs) were identified, demonstrating the presence of two alternative promoters that we termed pα and pβ. Whereas the 5′UTR originating from pα, was split up into three exons, a single exon represented the 5′ UTR of pβ transcripts. Strikingly, in heart, skeletal muscle and tonsils the upstream promoter pα was totally inactive and ART3 transcription appears to be driven solely by pβ. In all other cell types tested, transcription started mainly (if not exclusively) at pα. Thus, ART3 expression in human cells appears to be governed by a combination of differential splicing and tissue-preferential use of two alternative promoters. This specific use is evolutionary conserved as shown by analysis of the 5′ UTR of the mouse ART3 mRNA.

Keywords: Alternative splicing; Inverse 5′ RACE-PCR; 5′UTR; Alternative promotor; Mono ADP-ribosyltransferase

The Rab11-FIP1/RCP gene codes for multiple protein transcripts related to the plasma membrane recycling system by Min Jin; James R. Goldenring (pp. 281-295).

Rab11a is a member of the Rab11 small GTPase family, and plays an important role in plasma membrane recycling. Rab11-Family Interacting Protein 1 (Rab11-FIP1) binds to Rab11 through a carboxyl-terminal amphipathic alpha helix. We have identified eight alternatively spliced Rab11-FIP1 gene transcripts from human chromosome 8. Among them, Rab11-FIP1A-D have carboxyl terminal Rab11 binding domains, while Rab11-FIP1E-H do not contain the Rab11 binding domain. While Rab11-FIP1B and F gene transcripts are ubiquitous, other Rab11-FIP1 transcripts demonstrate more limited patterns of expression in human tissue cDNAs. EGFP-Rab11-FIP1A-D proteins over-expressed in HeLa cells targeted to Rab11a-containing membranes, while EGFP-Rab11-FIP1E/F and H proteins did not localize with recycling system membranes. However, transferrin trafficking was not significantly altered in HeLa cells over-expressing expressing any of the EGFP-Rab11-FIP1 proteins. Rabbit polyclonal antibodies specific for Rab11-FIP1B and Rab11-FIP1C/RCP demonstrated that Rab11-FIP1B and Rab11-FIP1C/RCP are expressed endogenously. Strikingly, endogenous staining for Rab11-FIP1C/RCP only partially co-localized with EGFP-Rab11-FIP1A, EGFP-Rab11-FIP1B, and EGFP-Rab11a in the perinuclear region, indicating that Rab11-FIP1C/RCP resides in a differentiable subcellular compartment within the plasma membrane recycling system compared with Rab11-FIP1A and Rab11-FIP1B. These data suggest that Rab11-FIP1 proteins may play coordinated roles in regulating plasma membrane recycling with regional specificity within the Rab11a-containing recycling system.

Keywords: Abbreviations; Rab11-FIP; Rab11-family interacting protein; RCP; Rab coupling protein; MTC panel; multi-tissue cDNAs panel; TfnR; transferrin receptor; TF; transferrinRab11-FIP1; RCP; Splice variant; Tissue expression; Rab11 binding domain; Transferrin trafficking

Involvement of LuxR, a quorum sensing regulator in Vibrio harveyi, in the promotion of metabolic genes: argA, purM, lysE and rluA by Carol M. Miyamoto; Edward A. Meighen (pp. 296-307).

Quorum sensing, involving signal transduction via the two-component response regulator LuxO to its downstream target LuxR, controls luminescence in the marine bacterium Vibrio harveyi. LuxR is a DNA binding protein that acts as both activator of the lux operon and repressor of its own gene. In order to determine if any other genes are affected by quorum sensing in V. harveyi, an assay for luxR-dependent promotion was devised using a genomic library maintained in a novel luxAB (luciferase) reporter. Screening in Escherichia coli DH-21 ( lacI^sq) entailed the addition of a second plasmid containing luxR under plac control. Four out of 5000 colonies showed luminescence stimulation upon IPTG induction of luxR. The four luxR-dependent promoters were upstream of argA, purM, lysE, and rluA, genes involved in arginine and purine biosyntheses, amino acid efflux, and pseudouridine synthesis, respectively. Based on analysis of luxR-dependent promoters, particularly that of argA, we describe a LuxR binding site, and implicate the coordination of LuxR with ArgR.

Keywords: Luminescence; Vibrio; V. harveyi; LuxR; ARG box; Quorum sensing

Directed evolution of plant basic helix–loop–helix transcription factors for the improvement of transactivational properties by Sitakanta Pattanaik; Claire H. Xie; Que Kong; Katherine A. Shen; Ling Yuan (pp. 308-318).

Myc-RP from Perilla frutescens and Delila from Antirrhinum majus, two plant basic helix–loop–helix transcription factors (bHLH TFs) involved in the flavonoid biosynthetic pathway, have been used for the improvement of transactivational properties by directed evolution. Through two rounds of DNA shuffling, Myc-RP variants with up to 70-fold increase in transcriptional activities have been identified using a yeast transactivation system. In a tobacco protoplast transient expression assay, one of the most improved variants, M2-1, also shows significant increase of transactivation. The majority of resulting mutations (∼53%) are localized in the acidic (activation) domains of the improved Myc-RP variants. In variant M2-1, three of the four mutations (L301P/N354D/S401F) are in the acidic domain. The fourth mutation (K157M) is localized to a helix within the N-terminal interaction domain. Combinatorial site-directed mutagenesis reveals that, while the acidic domain mutations contribute modestly to the increase in activity, the K157M substitution is responsible for 80% of the improvement observed in variant M2-1. The transactivation activity of the K157M/N354D double mutant is equal to that of M2-1. These results suggest that the interaction domain plays a critical role in transactivation of these bHLH TFs. Delila variants have also been screened for increased activities toward the Arabidopsis chalcone synthase ( CHS) promoter, a pathway promoter that responds weakly to the bHLH TFs. Variants with increased activity on the CHS promoter, while maintaining wildtype-level activities on the naturally responsive dihydroflavonol reductase promoter, have been obtained. This study demonstrates that functional properties of TFs can be modified by directed evolution.

Keywords: Plant transcription factors; Basic helix–loop–helix transcription activators; Transactivation; Acidic domain; Interaction domain; Directed evolution

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BBA - Gene Structure and Expression (v.1759, #6)