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BBA - Gene Structure and Expression (v.1759, #5)

Editorial Board (pp. ii).

Molecular properties and intracellular localization of rat liver nuclear scaffold protein P130 by Yasuhide Hibino; Tatsuhiro Usui; Yasuhiro Morita; Noriko Hirose; Mari Okazaki; Nobuhiko Sugano; Koichi Hiraga (pp. 195-207).

We examined the molecular basis of rat P130, a nuclear scaffold protein, and its functions. P130 comprising 845 amino acid residues possesses several functional domains and yields an electrophoretically distinctive isoform, P123, by altering its phosphorylation status in association with translocation across the nuclear membrane and from the digitonin-extractable fraction of the nucleus to the nuclear scaffold. The functional domains, NLS, NES, and zinc-finger bearing DNA-binding domains, ZF1 and ZF2, aid these translocations. P130 binds RNA through two RNA-binding domains (RB1 and RB2) similar to those of hnRNPs I and L. Microsome- and polysome-localized P130 and P123 were found in rat liver and Ac2F hepatoma cells. This localization required prior entry of P130 to the nucleus, but did not require RB1 and RB2. Thus, P130 initially purified from rat liver nuclear scaffold has the potential to play a variety of roles in biological events not only in the nuclear scaffold but also in various subcellular compartments. P130 (AB205483) is identical to matrin 3 (M63485 and BC062231), although the primary structure of rat matrin 3 has been revised, since it was first published.

Keywords: Nuclear scaffold; Matrin 3; Protein phosphorylation

Characterization of human cytoglobin gene promoter region by XiuMei Guo; Sjaak Philipsen; Kian-Cheng Tan-Un (pp. 208-215).

Cytoglobin (CYGB) is a member of the vertebrate globin family together with hemoglobin, myoglobin and neuroglobin. Although the physiological function of CYGB is still unclear, spectroscopic studies show that CYGB contains a hexacoordinated heme pocket similar to other pentacoordinated globin proteins. CYGB shares a common phylogenetic ancestry with vertebrate myoglobin from which it diverged by duplication before the appearance of jawed vertebrates. The objective of this study is to identify the regulatory and promoter region of the human cytoglobin gene. 5′ unidirectional deletion constructs demonstrated that the proximal promoter elements of human CYGB gene are located between −1113 to −10 relative to the translation start site. Site-directed mutagenesis showed that mutation of a c-Ets-1 motif at −1008 and Sp1 motifs at −400, −230 and −210 remarkably decreased the promoter activity. Gel shift assays confirmed the binding of DNA-nuclear proteins to these motifs. All these results indicate that CYGB gene expression can be up-regulated by c-Ets-1 and Sp1 motifs.

Keywords: Cytoglobin (CYGB); Promoter region; Transcription factor; c-Ets-1; Sp1

A novel Xenopus laevis larval keratin gene, xlk2: Its gene structure and expression during regeneration and metamorphosis of limb and tail by Ichiro Tazawa; Keiko Shimizu-Nishikawa; Katsutoshi Yoshizato (pp. 216-224).

A novel cytokeratin (CK) gene, xlk2, was cloned from a cDNA library prepared from regenerating limbs of Xenopus larvae. The deduced amino acid sequence indicated that its product, XLK2, is a 48 kDa type I (acidic) CK and has a high similarity to CK13, 15, and 19 with the highest homology (58%) to mouse CK15. The gene of xlk2 exclusively expressed in basal cells of the bi-layered larval epidermis, but not in other cells in larvae and not in other periods of life. Its expression was down-regulated during spontaneous and thyroid hormone-induced metamorphosis. The basal cells of the apical epidermal cap (AEC) formed on the regenerate of larval limbs terminated the expression of xlk2, whereas those of the adjacent normal epidermis continued to express it. The AEC-basal cells did not re-express the gene in the regenerate. In contrast, the basal cells of the tail regenerate also once terminated the expression of xlk2, but was able to re-express xlk2 later, supporting a notion that the “de-differentiated�? basal cells of the tail epidermal regenerate re-differentiate into larval normal epidermal cells.

Keywords: Thyroid hormone; Epidermis; Subtractive gene screening; Apical epidermal cap; Blastema; Basal cell

Identification and expression of a new splicing variant of FAD-sulfhydryl oxidase in adult rat brain by Jean Radom; Didier Colin; Franck Thiebault; Mai Dognin-Bergeret; Georges Mairet-Coello; Annick Esnard-Feve; Dominique Fellmann; Michèle Jouvenot (pp. 225-233).

Flavoproteins of the quiescin/sulfhydryl oxidase (QSOX) family catalyze oxidation of peptide and protein thiols to disulfides with the reduction of oxygen to hydrogen peroxide. We report here the molecular cloning of a new putative sulfhydryl oxidase cDNA, rQSOX-L (GenBank Accession noAY623665), from adult rat brain and its expression studied by RT-PCR, Northern and Western blots in rat tissues. DNA-sequencing demonstrated the existence of two cDNAs in rat cortex, corresponding to a long transcript ( rQSOX-L) and a short transcript ( rQSOX-S) which differed by 851 nucleotides due to alternative splicing. The new transcript, rQSOX-L (3356 nucleotides), was specifically expressed in brain, hypophysis, heart, testis and seminal vesicle. The distribution of this variant is not homogeneous in the different tissues studied and suggests a complex gene regulation. The full-length rQSOX-L cDNA has an open reading frame of 2250-bp encoding a protein of 750 amino acids that contains a signal peptide sequence, a protein-disulfide-isomerase-type thioredoxin and ERV1-ALR domains and a long form specific C-terminal extension. The rQSOX-L protein is highly homologous to members of the sulfhydryl oxidase/Quiescin family and contains particularly two potential sites for N-glycosylation. This protein isoform was specifically detected in rat brain tissues in opposition to the low molecular form that was ubiquitous. Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis of the immunoprecipitate tryptic fragments allowed the identification of rQSOX-L protein.

Keywords: Abbreviations; ALR; augmenter of liver regeneration; CBB; Coomassie brilliant blue; ERV1; gene essential for respiration and vegetative growth; MALDI-TOF MS; matrix-assisted laser-desorption/ionization-time-of-flight mass spectrometry; PDI; protein disulfide isomerase; QSOX; quiescin sulfhydryl oxidase; rQSOX-L; rat; QSOX; long transcript; rQSOX-S; rat; QSOX; short transcriptrQSOX-L sulfhydryl oxidase; Rat adult brain; Alternative splicing; Genomic organization; Expression pattern; MALDI-TOF MS

Characterization of the MCT-1 pseudogene: Identification and implication of its location in a highly amplified region of chromosome 20 by Suvobroto Nandi; Bo Shi; Jonathan Perreault; Ronald B. Gartenhaus (pp. 234-239).

The MCT-1 oncogene was initially identified as an amplified gene on chromosome Xq22–24 in a T-cell lymphoma. MCT-1 is over-expressed in a subset of diffuse large B-cell lymphoma (DLBCL), a common form of Non-Hodgkin's Lymphoma (NHL). We have identified a pseudogene for MCT-1 (ΨMCT-1) that is located on chromosome 20q11.2, a region within an amplicon containing several important genes frequently amplified in certain breast and ovarian cancers. Genomic analysis revealed that ΨMCT-1 is a processed pseudogene. Interestingly, both MCT-1 and its pseudogene are located on regions of the genome that are frequently amplified in several different human malignancies. MCT-1 is the oldest known oncogene and its insertion as a pseudogene occurred at a later time point in evolution. Existence of ΨMCT-1 should be considered when analyzing genomic amplification and or expression of MCT-1. Analysis of MCT-1 and ΨMCT-1 might provide clues to cancer genes and their evolution across species.

Keywords: Lymphoma; Pseudogene; MCT-1 oncogene; Gene amplification

Partial characterization of the mouse α-sarcoglycan promoter and its responsiveness to MyoD by Paul Delgado-Olguín; Félix Recillas-Targa; Haydeé Rosas-Vargas; Fabio Salamanca; Ramón M. Coral-Vázquez (pp. 240-246).

The mouse α-sarcoglycan gene is expressed in muscle cells during differentiation, but its transcriptional regulation is not understood. We have characterized the promoter region of the mouse α-sarcoglycan gene. This region is composed of positive and negative regulatory elements that respond to the myogenic differentiation environment. Accordingly, MyoD transactivates the α-sarcoglycan full-length and the proximal promoter. Chromatin immunoprecipitation assays revealed that MyoD, TFIID, and TFIIB interact with the distal promoter in C2C12 myoblasts, a stage at which the α-SG promoter appears to drive basal activity. In myotubes, such factors are located concomitantly at the distal promoter and at a DNA region around the proximal promoter. In agreement with these results, TFIID and TFIIB co-immunoprecipitate with MyoD. We conclude that the α-SG promoter is activated by MyoD, which interacts with TFIID and TFIIB in a protein complex differentially located at the distal promoter and around the proximal promoter during myogenic cell differentiation.

Keywords: α-Sarcoglycan promoter; MyoD transactivation; Core promoter

The role of Sp1 and Sp3 in the constitutive DPYD gene expression by Xue Zhang; Lin Li; Jeanne Fourie; James R. Davie; Vincenzo Guarcello; Robert B. Diasio (pp. 247-256).

Dihydropyrimidine dehydrogenase (DPD), the initial and rate-limiting enzyme in the 5-fluorouracil (5-FU) catabolic pathway, has been implicated as one of the factors determining the efficacy and toxicity of the anticancer agent 5-FU. Studies have attributed variation in DPD activity partially to alterations at the transcriptional level of DPYD gene. We investigated the transcription factors implicated in the constitutive expression of DPYD by utilizing a 174-bp fragment of the DPYD promoter region in which three consensus Sp protein binding sites (SpA, SpB and SpC) were predicted. The binding of Sp1 and Sp3 transcription factors to this region was detected by electrophoretic mobility shift and chromatin immunoprecipitation assays. By ectopically expressing human Sp1 and Sp3 in Sp-deficient Drosophila S2 cells, we demonstrated that Sp1 is a strong activator, while Sp3 by its own is a weak activator of the DPYD promoter. Moreover, Sp3 may serve as a competitor of Sp1, thus decreasing the Sp1 induced promoter activity. SpA, SpB and SpC sites are all Sp1 inducible. In the full activation of the DPYD promoter in human cell lines, the SpB site is essential; the SpC site works cooperatively with SpB, while SpA has minor promoter activity. These studies provide further insight into the molecular mechanisms underlying the heterogeneity of DPD activity, and may facilitate the efficacy and safety of 5-FU-based chemotherapy.

Keywords: Sp1; DPYD; Dihydropyrimidine dehydrogenase; Promoter

Promoter characterization and transcriptional regulation of Ncb5or, a novel reductase necessary for pancreatic β-cell maintenance by Kevin Larade; H. Franklin Bunn (pp. 257-262).

Ncb5or is a ubiquitously expressed gene required for β-cell survival in mice. Examination of mouse tissues demonstrated high levels of expression in the pancreas, heart and kidney. A transcription start site was identified 149 bp upstream from the start codon and transient expression analysis in βTC3 cells indicated the presence of a core promoter located within 348 bp upstream of this site. Deletion of Region C (−216/−157) resulted in a significant decrease in promoter activity and specific nucleotides located in a region designated C2 were demonstrated to be critical for complex binding. Deletion of Region D (−60/−33), which contains multiple consensus Sp1 sites, resulted in an additional loss of promoter activity. The data presented here identify and characterize the previously unknown promoter of Ncb5or, a reductase critical for β-cell survival.

Keywords: Abbreviations; EMSA; electrophoretic mobility shift assays; MODY; M; aturity; O; nset; D; iabetes in the; Y; oung; Ncb5or; N; -terminal; c; ytochrome; b5; domain; +; cytochrome b5; o; xido; r; eductase domain gene; TF; transcription factor; TSS; transcription start siteNcb5or; Reductase; Promoter; Beta cell; Diabetes

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BBA - Gene Structure and Expression (v.1759, #5)