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BBA - Gene Structure and Expression (v.1730, #2)

Editorial Board (pp. ii).

Characterization of O-methyltransferase ScOMT1 cloned from Streptomyces coelicolor A3(2) by Youngdae Yoon; Yong Sub Yi; Youngshim Lee; Seunghyun Kim; Bong Gyu Kim; Joong-Hoon Ahn; Yoongho Lim (pp. 85-95).

The roles of O-methyltransferases (OMTs) in microorganisms are not well understood, and are suggested to increase antimicrobial activity. Studies on OMTs cloned from microorganisms may help elucidate their roles. Streptomyces coelicolor A3(2) produces many useful natural antibiotics such as actinorhodin. Based on sequence information from S. coelicolor A3(2) genome, it was possible to clone several methyltransferases. An OMT cloned from Streptomyces coelicolor A3(2), ScOMT1 was characterized by in vivo and in vitro assays. Of 23 compounds tested, 13 were found to serve as its substrates. Of the 13 substrates, the methylated positions of 7 compounds were determined by HPLC, NMR, and MS analyses. This OMT favored ortho-dihydroxyflavones. Among the compounds tested here, the best substrate is 6,7-dihydroxyflavone.

Keywords: O; -methyltransferase; Streptomyces coelicolor; A3(2); Flavonoid

Identification and characterization of membralin, a novel tumor-associated gene, in ovarian carcinoma by Yu-Chi Chen; Ben Davidson; Chih-Chien Cheng; Anirban Maitra; Robert L. Giuntoli II; Ralph H. Hruban; Tian-Li Wang; Ie-Ming Shih (pp. 96-102).

Identification of new genes in cancer is the key to understand the molecular basis of tumor development as well as provide potential diagnostic markers and therapeutic targets. A novel gene, membralin (GeneBank accession number:DQ005958), was cloned from a human ovarian cancer cell line. Human membralin is unique and does not share significant sequence homology with other human genes, only membralins of other species. The gene contains 11 exons which encode at least two spliced variants in human cancer. The long form of membralin (membralin-1) comprises all 11 exons, encoding a protein of 620-amino acids long and the short form of membralin (membralin-3) contains all exons except for exon 10, encoding a protein of 408 amino acids. Expression of different membralin isoforms depends on tissue type. The long form, membralin-1, is expressed in ovarian and colorectal carcinomas but not in breast or pancreatic carcinomas, which express only the short splice form, membralin-3. Membralin-1–GFP fusion protein demonstrates exclusive cytoplasmic localization. Based on quantitative real-time PCR, in situ hybridization and Western blot analysis, membralin was highly expressed in ovarian serous carcinomas as compared to ovarian surface epithelium ( P<0.001). Ovarian carcinomas in effusions demonstrated a significantly higher level of membralin expression than in solid tumors ( P<0.001). In conclusion, these findings represent the first characterization of human membralin and suggest that membralin is a novel tumor-associated marker in ovarian serous carcinomas.

Keywords: Membralin; Tumor; Carcinoma

GhHb1: A nonsymbiotic hemoglobin gene of cotton responsive to infection by Verticillium dahliae by Zhan-Liang Qu; Hai-Yun Wang; Gui-Xian Xia (pp. 103-113).

Verticillium wilt of cotton is a widespread and destructive disease that is caused by the fungus pathogen Verticillium dahliae. Although no cotton cultivar is immune to the disease, some genotypes exhibit superior wilt tolerance. To gain an insight into the molecular mechanisms responsible for wilt tolerance, we employed the method of suppression subtractive hybridization (SSH) to isolate genes whose expression is up-regulated after inoculation of the pathogen in a wilt-tolerant cotton cultivar ( Gossypium hirsutum cv. BD18). Among the identified candidate ESTs, a cDNA representing a nonsymbiotic hemoglobin gene (designated GhHb1) was further characterized in this study. Northern blot hybridization demonstrated that GhHb1 shares similar characteristics to some other nonsymbiotic hemoglobin genes including the hypoxic stress-induced expression. Sub-cellular localization analysis indicated that GhHb1 proteins were predominantly present in the nucleus with a minor amount appearing in the cytoplasm. Two novel features of GhHb1 were also identified, indicating that GhHb1 expression is activated in the cotton roots after inoculation with V. dahliae and that exogenous hydrogen peroxide induces GhHb1 expression. These results suggest that the GhHb1 may play a role in the defense response of G. hirsutum against V. dahliae invasion.

Keywords: GhHb1; Hemoglobin; Verticillium dahliae

Wheat non-specific lipid transfer protein genes display a complex pattern of expression in developing seeds by Freddy Boutrot; Anne Guirao; R�mi Alary; Philippe Joudrier; Marie-Fran�oise Gautier (pp. 114-125).

Nine cDNA clones encoding non-specific lipid transfer proteins (nsLTPs) were isolated from Triticum aestivum and Triticum durum cDNA libraries and characterized. One cDNA is predicted to encode a type 2 nsLTP (7 kDa) while others encode type 1 nsLTPs (9 kDa). All encoded proteins contain an N-terminal signal sequence and possess the characteristic features of nsLTPs. The genomic structures of the wheat nsLtp genes show that type 2 TaLtp7.1a, TaLtp7.2a and type 1 TaLtp9.2b genes lack introns while the other type 1 genes consist of one intron. Construction of a phylogenic tree of Poaceae nsLTPs shows that wheat nsLTPs can be divided into eleven distinct groups and are closely related to barley sequences. Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, the expression patterns of nine nsLtp genes were studied during wheat seed development and germination. We identified three different profiles of nsLtp gene transcript accumulation. Whereas TdLtp7.1a, TdLtp9.4a and TdLtp9.7a transcripts were detected during all maturation stages, TdLtp7.2a, TdLtp9.2a, TdLtp9.3a, TdLtp9.5a and TdLtp9.6a transcripts were only present in the first and TdLtp9.1a in the last stages of seed development. Moreover, these nine wheat nsLtp genes are not seed-specific and are also expressed in the coleoptile of young seedlings. The present study revealed the complexity of the wheat nsLtp gene family and showed that the expression of nsLtp genes is developmentally regulated in the seeds, suggesting a specific function for each of the corresponding proteins.

Keywords: Evolution; Gene expression; nsLtp; gene; RT-PCR; Seed development; Wheat

Characterization of the 5′-flanking region of the human transcription factor Sp3 gene by Alicia Tapias; Paloma Monasterio; Carlos J. Ciudad; V�ronique No� (pp. 126-136).

A fragment of 1079 bp from the 5′-flanking region of the human Sp3 gene was isolated and characterized. The Sp3 promoter is a GC-rich region that contains putative binding sites for Elk-1, c-Myb, NF-1, Ap1, Sp1, NF-Y, Ap2 and USF. Several transcriptional start sites located between 70 and 132 bp upstream of the translational start site were identified. The proximal promoter was contained in the first 281 bp 5′ of the translational start, whereas the region including up to −225 relative to the translational start was referred as the minimal promoter. Transient transfections and luciferase assays revealed activation of the Sp3 proximal promoter upon overexpression of either Sp1 or Sp3, alone or in combination. Gel-shift and supershift assays demonstrated specific binding of Sp1 and Sp3 proteins to the GC box located in the proximal promoter of Sp3. Overexpression of NF-YA had a synergistic effect on Sp1 overexpression and an additive effect on Sp3 overexpression. Additionally, overexpression of NF-YA, Sp1 and Sp3 altogether had a synergistic effect on Sp3 promoter activity. Furthermore, binding of the NF-Y complex to the CCAAT box located in the proximal promoter of Sp3 was observed in gel-shift assays.

Keywords: Sp3; Promoter; Sp1; NF-Y; Gene regulation; Transcription

Genomic organization and functional analysis of the gene encoding the Kr�ppel-like transcription factor KLF6 by Ricardo C. Gehrau; Diego S. D'Astolfo; Claudio Prieto; Jos� L. Bocco; Nicol�s P. Koritschoner (pp. 137-146).

The Krüppel-like transcription Factor 6 (KLF6) is regulated during cell proliferation and differentiation events like mammalian development and tissue regeneration, while its aberrant expression is associated with tumor formation. To investigate KLF6 transcriptional control, the genomic organization of human KLF6 together with its cis-regulatory region was analyzed. A high sequence homology of KLF6 regulatory regions was found in mammals, which in turn predicts a high degree of evolutionary conserved transcriptional mechanisms. A transcription start site was identified at the first nucleotide downstream of a potential initiator element. Also, the role of KLF6 regulatory regions was determined by transfection experiments. A minimal promoter region lacking a TATA-box yet containing an Initiator was identified and found to be active in all cells analyzed. In addition, two strong activating sequences were located between positions −407/−344 and −307/−207, where the latter contained Sp1 and CAAT-box sites. Furthermore, ectopic expression of Sp1 increased the transcriptional activity of the KLF6 promoter. In conclusion, our data revealed that KLF6 gene transcription is under control of a TATA-box independent initiation mechanism together with an evolutionary conserved array of positive cis-acting elements.

Keywords: Gene expression; Transcription; Promoter; cis-acting element; Kr�ppel-like transcription factor; KLF6; Sp1

Functional characterization of the promoter of the human glucose transporter 10 gene by Fernando Segade; Dax C. Allred; Donald W. Bowden (pp. 147-158).

The human SLC2A10 gene encodes the high-affinity glucose transporter 10 (GLUT10) and is widely expressed in adult tissues, including organs which play major roles in glucose homeostasis. Its function and genomic location in a region linked to Type 2 diabetes susceptibility are consistent with a potential role in Type 2 diabetes. Analysis of the CpG-rich promoter revealed the presence of two major transcription start points with differential use in tissues and cell lines. Mapping of transcriptionally active regions in the 5′ flanking sequence identified a region, located between nucleotides −70 and −14 (relative to the major transcription start point) as the SLC2A10 basal promoter. This sequence harbors consensus binding sites for Sp, AP2α, and other transcription factors. A juxtaposed Sp/AP2α motif located between −25 and −11 is critical for core promoter function. In cells expressing Sp and AP2 factors, the two motifs are required for maximal activation of the basal promoter. In cells lacking AP2α, transcription is dependent on the integrity of the Sp site. Using electrophoresis mobility shift assays, we demonstrate that Sp1 and Sp3 bind to the GC-box in site 5 forming specific complexes. In addition, a silencer region is present upstream of −696 which down-regulates SLC2A10 promoter activity independently of its distance to the transcript start site.

Keywords: Abbreviations; GLUT; glucose transporter; PAC; P1 artificial chromosome; TSP; transcription start point; PCR; polymerase chain reaction; UTR; untranslated region; EMSA; electrophoretic mobility shift assay; RACE; rapid amplification of cDNA ends; T2DM; type 2 diabetes mellitusGLUT10; Sp1; AP2; Silencer; Diabetes; Overlapping motif

Functional analysis of fengycin synthetase FenD by Tsuey-Pin Lin; Chyi-Liang Chen; Hui-Chuan Fu; Cheng-Yeu Wu; Guang-Huey Lin; Shih-Hao Huang; Li-Kwan Chang; Shih-Tung Liu (pp. 159-164).

Fengycin is a cyclic lipopeptidic antibiotic produced nonribosomally by Bacillus subtilis. A fengycin synthetase mutant of B. subtilis F29-3 was generated with Tn 917lux, which contains a transposon inserted in a 7716-bp gene, fenD. The mutation can be genetically complemented by transforming a plasmid carrying a wild-type fenD, confirming the participation of the gene in fengycin synthesis. Sequencing and biochemical analysis reveal that this gene encodes an enzyme that includes two amino acid-activating modules, FenD1 and FenD2, which activatel-Tyr andl-Thr, the third and the fourth amino acids in fengycin, respectively.

Keywords: Fengycin; Peptide synthetase; Nonribosomal peptide synthesis

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BBA - Gene Structure and Expression (v.1730, #2)