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BBA - Gene Structure and Expression (v.1729, #2)

Editorial Board (pp. ii).

The 5′ region of the human hSUV3 gene encoding mitochondrial DNA and RNA helicase: Promoter characterization and alternative pre-mRNA splicing by Michal Minczuk; Joanna Lilpop; Joanna Boros; Piotr P. Stepien (pp. 81-87).

The human nuclear hSUV3 gene encodes ATP-dependent RNA and DNA helicase, which predominantly localizes in the mitochondria. In yeast, the Suv3 helicase is a component of mitochondrial degradosome, a two-subunit complex, which degrades aberrant mtRNAs. In contrast to the well-documented physiological role of the yeast SUV3, the function of its human orthologue remains unknown. In this report, we have analyzed the hSUV3 5′ genomic region. Our data suggest that hSUV3 is a housekeeping gene. Deletion analysis and in vitro mutagenesis revealed the presence of an enhancer region and regulatory elements in basal promoter including: (i) direct 10-bp-long repeats, which share significant sequence similarity with the consensus for the NF-κB/Rel family transcription factors, (ii) Sp1 general transcription factor binding site, and (iii) NRF-1 transcription factor binding sites, the latter typical for nuclear-encoded mitochondrial genes. Furthermore, we show that the 5′ region of the hSUV3 pre-mRNA can be alternatively spliced.

Keywords: Housekeeping gene; Alternative splicing; Transcription factor; Promoter analysis; Reporter assay

Inhibition of NF-κB activation and expression of inflammatory mediators by polyacetylene spiroketals from Plagius flosculosus by Marco A. Calzado; Katharina Schmid L�di; Bernd L. Fiebich; Yinon Ben-Neriah; Susanne Bacher; Eduardo Munoz; Mauro Ballero; Simona Prosperini; Giovanni Appendino; M. Lienhard Schmitz (pp. 88-93).

Transcription factor NF-κB plays a key role for the inducible expression of genes mediating proinflammatory effects and is thus an important target for the development of antiinflammatory drugs. Here, we show that extracts from the plant Plagius flosculosus (L.) Alavi and Heyw. can inhibit the induction of NF-κB activity, and we describe the identification of three spiroketal compounds. Of those, only compound 1 could inhibit the phosphorylation and proteasomal degradation of IκB, thus preventing the nuclear import and DNA binding of NF-κB. Accordingly, compound 1, which is also found in the widely used medicinal herb chamomile, interfered with the LPS-induced production of IL-1, IL-6, TNF, and PGE2 in primary human monocytes.

Keywords: NF-κB; Inflammation; Cytokine; Inducible transcription

Multiple proteins are involved in the protein–DNA complex in the proximal promoter of the human α1(III) collagen gene ( COL3A1) by Tomoaki Yoshino; Hideaki Sumiyoshi; Toshitaka Shin; Noritaka Matsuo; Yutaka Inagaki; Yoshifumi Ninomiya; Hidekatsu Yoshioka (pp. 94-104).

We have characterized the proximal promoter of the human α1(III) collagen gene ( COL3A1). Transient transfection assays using a series of chimeric constructs linked to the luciferase gene indicated that the segment from −96 to −34 is necessary to activate transcription. Electrophoretic mobility shift assays (EMSAs) showed that the multiple proteins form the DNA–protein complex in different combinations depending on the cell types. A competition assay using mutant oligonucleotides showed that the sequence 5′-GCTCTCATATTTCAGAA-3′ (−79 to −63 bp) is critical for DNA–protein complex formation. This sequence is contained in the B element of mouse α1(III) collagen gene ( Col3a1) reported by Ruteshouse and de Crombrugghe (J. Biol. Chem., 1993). In the rhabdomyosarcoma cell line, A204, at least two proteins of 92–118 kDa and 40–52 kDa are involved in the DNA–protein complex bound to this motif.

Keywords: Abbreviations; BBF; B element binding factor; AGPC; acid guanidium phenol chloroform; DMEM; Dulbecco's modified Eagle's medium; FBS; fetal bovine serum; vSMC; vascular smooth muscle cells; ssDNA; salmon sperm DNAType III collagen; Promoter; Transcription; DNA binding protein

Identification and characterisation of two runx2 homologues in zebrafish with different expression patterns by T. van der Meulen; S. Kranenbarg; H. Schipper; J. Samallo; J.L. van Leeuwen; H. Franssen (pp. 105-117).

Genome and gene duplications are considered to be the impetus to generate new genes, as the presence of multiple copies of a gene allows for paralogues to adopt novel function. After at least two rounds of genome/gene duplication, the Runt gene family consists of three members in vertebrates, instead of one in invertebrates. One of the family members, Runx2, plays a key role in the development of bone, a tissue that first occurs in vertebrates. The family has thus gained new gene function in the course of evolution. Two Runx2 genes were cloned in the vertebrate model system the zebrafish ( Danio rerio). The expression patterns of the two genes differ and their kinetics differ up to four fold. In addition, splice forms exist that are novel when compared with mammals. Together, these findings comprise opportunities for selection and retention of the paralogues towards divergent and possibly new function.

Keywords: Zebrafish; Runx2; Gene duplication; Differential expression; Splice form; Gene divergence

Isolation and expression analysis of genes encoding DNA methyltransferase in wheat ( Triticum aestivum L.) by Yan Dai; Zhongfu Ni; Jing Dai; Tao Zhao; Qixin Sun (pp. 118-125).

DNA methylation of cytosine residues, catalyzed by DNA methyltransferases, is suggested to play important roles in regulating gene expression and plant development. In this study, we isolated four wheat cDNA fragments and one cDNA with open reading frame encoding putative DNA methyltransferase and designated TaMET1, TaMET2a, TaMET2b, TaCMT, TaMET3, respectively. BLASTX searches and phylogenetic analysis suggested that five cDNAs belonged to four classes ( Dnmt1, Dnmt2, CMT and Dnmt3) of DNA methyltransferase genes. TaMET2a encoded a protein of 376 aa and contained eight of ten conserved motifs characteristic of DNA methyltransferase. Genomic sequence of TaMET2a was obtained and found to contain ten introns and eleven exons. The expression analysis of the five genes revealed that they were expressed in developing seed, during germination and various vegetative tissues, but in quite different abundance. It was interesting to note that TaMET1 and TaMET3 mRNAs were clearly detected in dry seeds. Moreover, the differential expression patterns of five genes were observed between wheat hybrid and its parents in leaf, stem and root of jointing stage, some were up-regulated while some others were down-regulated in the hybrid. We concluded that multiple wheat DNA methyltransferase genes were present and might play important roles in wheat growth and development.

Keywords: DNA methylation; Wheat; Methyltransferase gene; Expression; Heterosis

Transcriptional control of the human urothelial-specific gene, uroplakin Ia by G.D. Hall; R.J. Weeks; J. Olsburgh; J. Southgate; M.A. Knowles; P.J. Selby; J.D. Chester (pp. 126-134).

The transcriptional control elements of tissue-specific genes may be exploited in the design of therapeutic constructs for use in human gene therapy. The uroplakins are a family of four proteins which form the asymmetric unit membrane of the urothelium. We have cloned the human uroplakin Ia gene and defined its genomic structure and transcriptional start site. Using quantitative RT-PCR in an extended panel of normal tissues, we have demonstrated highly urothelial-specific expression of this gene. A Dual-Luciferase assay was used to assess the transcriptional activity of a variety of promoter fragments of the human uroplakin Ia gene. A highly specific promoter fragment (consisting of 2147 bp of 5′-flanking sequence, intron 1 and the 5′ UTR) was identified which regulated urothelial-specific expression in vitro. The human uroplakin Ia promoter identified has potential use in future gene therapy strategies to restrict transgene expression to the urothelium.

Keywords: Uroplakin 1a; Promoter; Tissue-specific; Urothelial; Gene therapy

Identification of a structural element that is essential for two functions of transcription factor NusG by Lislott V. Richardson; John P. Richardson (pp. 135-140).

The transcription factor NusG from Escherichia coli modulates the rate of transcript elongation by RNA polymerase and the efficiency of Rho-dependent transcript termination. It consists of two globular domains with an extra loop extending out of the amino-terminal domain in the position that is occupied by a third globular domain in some NusG homologues. We have tested the role of this appended mini-domain by assaying the elongation and termination enhancement activities of variants. The results show that variants with changes in their sequence do not cause a loss of functions, whereas variants with the deletions of the residues in that domain are much less active for both functions. This finding suggests that the mini-domain serves as a structural element for an interaction rather than as a site for residue-specific contacts.

Keywords: RNA polymerase; Transcript elongation; Transcript termination; Termination factor Rho

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BBA - Gene Structure and Expression (v.1729, #2)