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BBA - Gene Structure and Expression (v.1730, #1)

Editorial Board (pp. ii).

Genomic organisation and tissue specific expression of ABLIM2 gene in human, mouse and rat by Eugene Klimov; Olga Rud'ko; Elian Rakhmanaliev; Galina Sulimova (pp. 1-9).

The exon–intron structures of the human, rat and mouse ABLIM2 gene were determined in silico. The experimental verification resulted in the revealing of two mRNA isoforms of the ABLIM2 gene. The isoforms a and b contained 20 exons and 18 exons, respectively. The highest expression of both isoforms was observed in rat brain and eye and in mouse embryos. The 5′-UTR region of the ABLIM2 gene was 127 bp in rat and mouse, but in human, it was 65 bp. The site of polyadenylation was shown to be present at a distance of 682 bp from the stop-codon in human and rat and 684 bp in mouse. The in silico analysis of the gene 5′-region was performed. The high density of brain and CNS specific transcription factors' binding sites in the promoter region was shown for all three organisms. The comparison of the amino acid sequences of the human ABLIM2 and ABLIM1 proteins showed that the number and arrangement of domains (four LIM-domains in the N-end region and the C-end VHP-domain) were similar. The structure of the ABLIM2 proteins was similar in all three organisms. On the basis of our data, it was assumed that the ABLIM2 protein was necessary for the normal functioning of neurons.

Keywords: ABLIM2; gene; Exon–intron structure; Tissue specific expression; In silico promoter analysis

A novel Rel protein and shortened isoform that differentially regulate antibacterial peptide genes in the silkworm Bombyx mori by Hiromitsu Tanaka; Masafumi Yamamoto; Yuko Moriyama; Masafumi Yamao; Seiichi Furukawa; Aki Sagisaka; Hiroshi Nakazawa; Hajime Mori; Minoru Yamakawa (pp. 10-21).

Two cDNAs encoding novel Rel proteins were cloned from the silkworm, Bombyx mori. These cDNA clones ( BmRelA and BmRelB) showed identical nucleotide sequences except for the 5′-region. BmRelB cDNA derived probably from an alternatively spliced mRNA lacked 241 bp nucleotides at the 5′-region of the BmRelA cDNA, resulting in a loss of the first 52 amino acids. Expression of antibacterial peptide genes was strongly inhibited upon infection with Micrococcus luteus in transgenic silkworms in which BmRel gene expression was knocked down, suggesting that these two Rel proteins are involved in activation of antibacterial peptide genes. Co-transfection experiments indicated that BmRelB activated the Attacin gene strongly and other genes to a lesser extent, whereas BmRelA activated Lebocin 4 gene strongly and Attacin and Lebocin 3 genes very weakly. The Rel homology domain of BmRelA and BmRelB was shown to bind specifically to κB sites of antibacterial peptide genes. Proline-rich domains of the BmRels were necessary for activation of antibacterial peptide genes. These results illustrate that a minor structural change in Rel proteins can provoke a dramatic differential activation of antibacterial peptide genes, suggesting a novel regulatory mechanism for insect antibacterial peptide gene expression.

Keywords: Rel protein; Insect immunity; Antibacterial peptide; Transgenic silkworm; Gene knockdown; Differential transcriptional regulation

Discovery and characterization of new epididymis-specific beta-defensins in mice by Jenni Jalkanen; Ilpo Huhtaniemi; Matti Poutanen (pp. 22-30).

The male urogenital tract epithelium is exposed to several pathogens, but only a few are potent enough to cause infection in a healthy individual. The exact mechanisms that protect the male reproductive tract from ascending pathogenic micro-organisms are still poorly characterized. We recently reported a method to identify novel epididymis-specific genes by analyzing the expressed sequence tags (ESTs) present in the mouse epididymal cDNA library of the UniGene collection at National Center for Biotechnology Information (NCBI). In the present study, we discovered in silico two novel epididymal genes: the beta-defensins Defb41 and Defb42. The full-length cDNAs for the genes were acquired by the RT-PCR and 5′-RACE approaches and were subsequently sequenced. Q-RT-PCR and in situ hybridization revealed Defb41 and Defb42 to be expressed mainly in the proximal caput. The expression of both defensins was found to be regulated by androgens. Based on their structure and expression pattern, Defb41 and Defb42 are suggested to have a role in the antimicrobial protection of sperm and urogenital tract epithelia.

Keywords: Defensin; Epididymis; EST

Identification of functional candidate genes for body composition by expression analyses and evidencing impact by association analysis and mapping by Siriluck Ponsuksili; Eduard Murani; Karl Schellander; Manfred Schwerin; Klaus Wimmers (pp. 31-40).

This study aims to identify hepatic genes affecting traits related to muscularity and obesity by combining expression analyses, association studies, and gene mapping. Functional candidate genes with trait-associated expression were obtained by hybridising custom made application-specific cDNA microarrays with targets of discordant sib pairs of a porcine experimental population. Out of 238 genes addressed, nine genes were regulated by the factor ≥ 2 between the sib pairs. Differential gene expression was independently confirmed for selected genes by real time RT-PCR. Transcript levels of four genes (APOH, PEDF, SLCO1B3, TBG) were significantly different between the phenotype groups. Screening for trait associated markers within TBG and APOH by comparative sequencing of discordant sib pairs revealed a SNP at position nt 778 (A>C) (N229H) of TBG. No polymorphism in APOH was detected. Association analysis confirmed effects of TBG on carcass traits statistically. Allocating TBG to a QTL region on chromosome X revealed genetic evidence for the effect. Moreover, our results indicate that there are probably two polymorphisms segregating-one (N229H) altering binding capability of TBG and another still to be detected altering the transcription rate of TBG.

Keywords: Porcine cDNA microarray; SNP; Polymorphism; Association analysis; Thyroxine-binding globulin; Carcass trait; Obesity; Muscularity; Pig

Synergistic activation of the insulin gene promoter by the β-cell enriched transcription factors MafA, Beta2, and Pdx1 by Shinsaku Aramata; Song-iee Han; Kunio Yasuda; Kohsuke Kataoka (pp. 41-46).

Specific expression of the insulin gene in pancreatic islet β-cells requires multiple cis-regulatory elements in its promoter. Pdx1, MafA, and Beta2 have been identified as β-cell enriched transcription factors that bind to these elements. Pdx1 has been shown to bind to A1, A3, A5, and GG2, and Beta2 binds to E1 by forming a heterodimer with the ubiquitous factor E47. MafA was recently identified as a C1-element binding factor. However, interactions between these factors and the promoter have not been characterized in detail. In this report, we show that these transactivators synergistically stimulate insulin promoter activity. Among multiple binding sites for Pdx1, MafA, and Beta2, at least GG2, C1, and E1 elements located in the promoter region between −150 and −100 base pairs are necessary for the synergism. We also found that neither MafB nor c-Maf, close relatives of MafA, showed synergistic activation. These results suggest that co-expression and functional synergism of these β-cell enriched transactivators, MafA, Pdx1, and Beta2, are critical for establishing the β-cell-specific and efficient expression of the insulin gene.

Keywords: Transcriptional regulation; Insulin; Transcription factor; Tissue-specific gene expression

Porphyrins and porphines bind strongly and specifically to tRNA, precursor tRNA and to M1 RNA and inhibit the ribonuclease P ribozyme reaction by Yoshiaki Hori; Maria C. Rogert; Terumichi Tanaka; Yo Kikuchi; Elena V. Bichenkova; Amanda N. Wilton; Abdul Gbaj; Kenneth T. Douglas (pp. 47-55).

Porphyrins and porphines strongly inhibit the action of the RNA subunit of the Escherichia coli ribonuclease P (M1 RNA). Meso-tetrakis(N-methyl-pyridyl)porphine followed linear competitive kinetics with pre-tRNA^Gly1 from E. coli as variable substrate ( K_i 0.960 μM). Protoporphyrin IX showed linear competitive inhibition versus pre-tRNA^Gly1 from E. coli ( K_i 1.90 μM). Inhibition by meso-tetrakis[4-(trimethylammonio)phenyl]porphine versus pre-tRNA^Gly1 from E. coli followed non-competitive kinetics ( K_i 4.1 μM). The porphyrins bound directly to E. coli tRNA^Val, E. coli pre-tRNA^Gly1 and M1 RNA and dissociation constants for the 1:1 complexes were determined using fluorescence spectroscopy. Dissociation constants (μM) against E. coli tRNA^Val and E. coli pre-tRNA^Gly were: meso-tetrakis(N-methyl-pyridyl)porphine 1.21 and 0.170; meso-tetrakis[4-(trimethylammonio)phenyl]porphine, 0.107 and 0.293; protoporphyrin IX, 0.138 and 0.0819. For M1 RNA, dissociation constants were 32.8 nM for meso-tetrakis(N-methyl-pyridyl)porphine and 59.8 nM for meso-tetrakis[4-(trimethylammonio)phenyl]porphine and excitation and emission spectra indicate a binding mode with strong π-stacking of the porphine nucleus and base pairs in a rigid low-polarity environment. Part of the inhibition of ribonuclease P is from interaction with the pre-tRNA substrate, resulting from porphyrin binding to the D-loop/T-loop region which interfaces with M1 RNA during catalysis, and part from the porphyrin binding to the M1 RNA component.

Keywords: Abbreviations; DMSO; dimethyl sulfoxide; PPIX; Protoporphyrin IX; RNase P; ribonuclease P; TCPP; meso-tetrakis(4-carboxyphenyl)porphine; T4MPyP; meso-tetrakis(N-methyl-pyridyl)porphine; TMAP; meso-tetrakis[4-(trimethylammonio)phenyl]porphineTransfer RNA; Ribozyme inhibitors; Fluorescence binding studies; Inhibition kinetics

Comparative expression of five Lea Genes during wheat seed development and in response to abiotic stresses by real-time quantitative RT-PCR by Mohamed A. Ali-Benali; R�mi Alary; Philippe Joudrier; Marie-Fran�oise Gautier (pp. 56-65).

Gene expression profiles of group 2 (dehydrins) and group 4 Late embryogenesis abundant ( Lea) genes in developing seeds of Triticum durum and T. aestivum and in coleoptiles and coleorhizae of T. durum seedlings were monitored by real-time quantitative RT-PCR. The five genes exhibited clear differences in their accumulation pattern in wheat seed and in response to dehydration, low temperature, salinity and ABA. Td29b, Td16 and Td27e gene transcripts accumulate late in embryogenesis as expected for Lea genes, Td11 gene transcripts were present throughout seed development whereas no Td25a gene transcripts were detected in seeds. Drastic changes in the relative levels of Td29b, Td16, Td27e and Td11 transcripts occurred at the shift between the cell expansion and desiccation phases. All genes except the Td11 gene are more highly induced by dehydration in coleorhizae than in coleoptiles. In contrast, response to low temperature, salinity or ABA is higher in coleoptiles than in coleorhizae. Depending on both the gene and on the type of stress, a wide range of induction levels (8- to 100,000-fold) was observed.

Keywords: Real-time quantitative PCR; Gene expression; Wheat; Seed development; Abiotic stresses; LEA proteins

Promoter characterization of Semaphorin SEMA3F, a tumor suppressor gene by Sophie Kusy; Vincent Potiron; Chan Zeng; Wilbur Franklin; Elisabeth Brambilla; John Minna; Harry A. Drabkin; Jo�lle Roche (pp. 66-76).

The tumor suppressor gene, Semaphorin SEMA3F, is frequently downregulated in lung cancer. Understanding the specific mechanism of SEMA3F suppression should be informative in terms of epithelial carcinogenesis and potential therapeutic interventions. Although a CpG-island is located 5083–3927 nt upstream of the translation start site, there have been no previous reports dealing with SEMA3F promoter regulation. We have now mapped the transcriptional initiation sites within the CpG-island and defined the region necessary for transcriptional activation. We then looked for evidence of SEMA3F promoter methylation since SEMA3F mutations are rare. By Southern blot and methylation-specific PCR assays, we identified a region in cell lines (i.e., area d at position minus 3850–3644 nt) for which methylation was significantly ( P<0.0001) correlated with loss of expression. However, histone deacetylase inhibition with Trichostatin A was much more effective than 5-aza-2′-deoxycytidine in stimulating SEMA3F. Our results suggest that while SEMA3F promoter methylation correlates with repression, chromatin remodeling through histone deacetylase inhibition is sufficient to activate SEMA3F expression.

Keywords: Lung cancer; Semaphorin SEMA3F promoter; Transcriptional initiation site; Reporter gene; Epigenetic modifications

Cloning and characterization of the human and rabbit NUDEL-oligopeptidase promoters and their negative regulation by Juliano R. Guerreiro; Sheila M.B. Winnischofer; Marta F. Bastos; Fernanda C.V. Portaro; Mari C. Sogayar; Antonio C.M. de Camargo; Mirian A.F. Hayashi (pp. 77-84).

NUDEL-oligopeptidase is a cytosolic cysteine peptidase, active towards oligopeptides and involved in the conversion and inactivation of a number of bioactive peptides. This protein interacts with neuronal proteins and is essential for brain development and cortical organization during embryogenesis. In this study, 5′-flanking sequences of the human and rabbit NUDEL-oligopeptidase gene were cloned into the pGL3 reporter gene vector and the promoter activity of the full-length fragment and deletions series was measured in transient transfection assays using two different cell lines, namely, C6 rat glioma and NH15 human neuroblastoma. Overall, a very similar pattern of promoter activity was obtained for both rabbit and human NUDEL-oligopeptidase promoter sequences, and their respective serial deletion constructs upon transient transfection into these cell lines. The only exception was for the longest rabbit upstream sequence that displayed about 1.8-fold higher luciferase expression upon transfection into NH15 neuronal cells than that observed upon transfection into C6 glioma cells. On the other hand, no significant difference was observed for the human longest sequence. These results are in good agreement with the expression pattern of NUDEL-oligopeptidase in human and rabbit tissues.

Keywords: Abbreviations; EOPA; endooligopeptidase A; NUDEL; NUclear Distribution Element-Like; r; NUDEL-oligopeptidase; recombinant NUDEL-oligopeptidase; α-; r; NUDEL-oligopeptidase; recombinant NUDEL-oligopeptidase antibody; Lis1; lissencephaly gene 1 product; DISC1; Disrupted-In-SChizophrenia; gene product; qf; quenched fluorescenceGene expression; Central nervous system; Tissue-specificity; Promoter regulation; Transcription factor

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BBA - Gene Structure and Expression (v.1730, #1)