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BBA - Gene Structure and Expression (v.1727, #3)

Editorial Board (pp. ii).

Molecular characterization of new members of the Hevea brasiliensis hevein multigene family and analysis of their promoter region in rice by Val�rie Pujade-Renaud; Christine Sanier; Laurence Cambillau; Arokiaraj Pappusamy; Heddwyn Jones; Natsuang Ruengsri; Didier Tharreau; Herv� Chrestin; Pascal Montoro; Jarunya Narangajavana (pp. 151-161).

The cloning of hevein genes from Hevea brasiliensis was undertaken with the objective to isolate useful promoters to drive transgene expression in genetically engineered rubber tree. Four different full length genes were cloned by library screening and a fifth, a partial gene, by adaptor-anchored PCR. Sequence alignment revealed that hevein genes, although highly conserved in their transcribed region, diverged in two groups, with major differences in their promoter region, suggesting a more rapid evolution of the upstream regulatory functions of the genes than the downstream functions of their protein products. The promoter regions from two hevein genes representative of each group were isolated and analyzed in rice. Although both were functional, only the longest promoter sequence (PHev2.1) conferred a high level of expression to the transgene in various tissues of this heterologous host. It was in addition up-regulated by mechanical wounding and fungal infection in leaves. A number of potential cis-regulatory elements were identified in silico and are discussed in view of the expression profiles observed in rice.

Keywords: Hevea brasiliensis; Hevein; Multigene family; Promoter; Biotic and abiotic stress; Rice

Cloning, sequencing and functional expression in Escherichia coli of the gene for a P-type Na⁺-ATPase of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum by Yuusuke Suzuki; Sumie Ueno; Rieko Ohnuma; Noriyuki Koyama (pp. 162-168).

Cloning and sequencing of the gene encoding a P-type Na⁺-ATPase of a facultatively anaerobic alkaliphile, Exiguobacterium aurantiacum, were conducted. The structural gene was composed of 2628 nucleotides. The deduced amino acid sequence (876 amino acid residues; Mr, 96,664) suggested that the enzyme possesses 10 membrane-spanning regions. When the amino acid sequences of the four putative membrane regions, M4, M5, M6 and M8, of BL77/1 ATPase were aligned with those of fungal Na⁺-ATPase, Na⁺/K⁺-ATPase, H⁺-ATPases and sarcoplasmic reticulum Ca²⁺-ATPase, it exhibited the highest homology with Ca²⁺-ATPase except M5 region. By the transformation of Escherichia coli with the expression vector (pQE30) containing the ATPase gene, the enzyme was functionally expressed in E. coli membranes.

Keywords: Alkaliphile; Na; ⁺; -ATPase; P-type ATPase

SNAMA, a novel protein with a DWNN domain and a RING finger-like motif: A possible role in apoptosis by Arshad Mather; Mpho Rakgotho; Monde Ntwasa (pp. 169-176).

We have characterized SNAMA a hitherto uncharacterized Drosophila protein that appears to play a role in apoptosis. SNAMA (something that sticks like glue) is a 1231 amino acid protein with a conserved 76 residue N-terminal domain called Domain With No Name (DWNN). The DWNN domain was first identified in cytotoxic T Cell-resistant CHO cells using promoter trap mutagenesis to screen for genes involved in apoptosis. Subsequently, this domain was identified in other eukaryotic organisms including animals and plants. The SNAMA transcript is abundant early in embryogenesis but reduced in older embryos and in adult males and females. Human and mouse homologues of SNAMA are known to bind to p53 and to the retinoblastoma protein (Rb) suggesting a role in transcriptional regulation and cell cycle control. We took advantage of a P-element insertion line in which the P-element is inserted in the first intron, to investigate the biological function of the gene. These mutants are lethal when homozygous. Apoptosis appears early during embryogenesis and is observed virtually throughout the gastrula. The DWNN domain has a ubiquitin-like fold and may interact with a subset of cellular proteins. There is also a conserved RING finger-like motif along the sequence of SNAMA following a C2HC zinc finger.

Keywords: Cell cycle; RNA processing; Retinoblastoma-binding protein (RBBP6); Retinoblastoma protein (Rb); p53

PAP IB, a new member of the Reg gene family: Cloning, expression, structural properties, and evolution by gene duplication by Emmanuelle Laurine; Xavier Manival; Claudine Montgelard; Chantal Bideau; Jean-Louis Berg�-Lefranc; Monique �rard; Jean-Michel Verdier (pp. 177-187).

Reg proteins are expressed in various organs and are involved in cancers and neurodegenerative diseases. They display a typical C-type lectin-like domain but possess additional highly conserved amino acids. By studying human databases and Expressed Sequence Tags library, we identified a new member called PAP IB. Using probabilistic approaches, we established a phylogenetic tree of eighteen Reg proteins. The dendogram showed that they constitute a superfamily composed of three distinct families (FI to FIII) of paralogues that resulted from duplication. We therefore focused on two proteins, REG Iα and PAP IB, belonging to the more closely related FI and FII families, respectively. REG Iα and PAP IB share 50% sequence identity. After cloning PAP IB, however, we found that it was expressed almost only in pancreas, unlike REG Iα, whose expression is ubiquitous. In addition, by building a model of the structure of PAP IB based on the X-ray structure of REG Iα, we observed that the two proteins displayed distinctive surface charge distribution, which may lead to different ligands binding. In spite of their common fold that should result in closely related functions, REG Iα and PAP IB are a good example of duplication and divergence, probably with the acquisition of new functions, thus participating in the evolution of the protein repertoire.

Keywords: Reg; Phylogeny; PAP IB; Homology modeling; Charge distribution; Structure–function relationship

Complementary DNA cloning, genomic characterization and expression analysis of a mammalian gene encoding histidine-rich calcium binding protein by Sunghee Hong; Tae-Wan Kim; Inchul Choi; Jong-Min Woo; Jungsu Oh; Woo Jin Park; Do Han Kim; Chunghee Cho (pp. 188-196).

A protein complex present at the junctional sarcoplasmic reticulum (SR) membrane is implicated in the Ca²⁺ release process during muscle contraction. The histidine-rich Ca²⁺-binding protein (HRC) is an emerging component associated into the SR protein complex. We cloned cDNAs for rat and monkey HRCs, showing a conserved sequence organization in common with other mammalian HRCs. Genomic analysis revealed that each mammalian HRC gene is present as a single copy in the genome, consisting of 6 exons and 5 introns. Developmental expression analysis using mouse embryos and postnatal hearts demonstrated that Hrc transcription begins at 12.5 days postcoitum and its level increases gradually, reaching an adult level in the range 5–20 days after birth. Comparing the Hrc gene and other SR genes, we found that the timing and pattern of gene expression vary among the SR genes and the full-level expression of these genes is achieved in the heart after postnatal day 20. Collectively, our study provides comprehensive information about the structure and expression of the mammalian HRC gene, together with the comparative expression data of the related SR genes.

Keywords: Cardiac muscle; Heart; Histidine-rich Ca; ²⁺; -binding protein; Mammal; Sarcoplasmic reticulum

The splicing factor U2AF65 is functionally conserved in the thermotolerant deep-sea worm Alvinella pompejana by Kristy L. Henscheid; David S. Shin; S. Craig Cary; J. Andrew Berglund (pp. 197-207).

Due to their inherent stability, thermophilic bacteria and archaea serve as important resources for biochemical and biophysical analyses of many biological processes. Unfortunately, scientists characterizing eukaryote-specific processes, such as nuclear pre-mRNA splicing, are unable to take advantage of these sources of thermostable proteins. To identify and provide a source of thermostable eukaryotic proteins, we are characterizing splicing factors in the thermotolerant deep-sea vent polychaete, Alvinella pompejana. This worm, also known as the Pompeii worm, is found in the extreme environment of deep-sea hydrothermal vents, and is one of the most thermotolerant eukaryotic organisms known. We report on detailed analyses of U2AF65, the large subunit of the U2 small nuclear ribonucleoprotein auxiliary factor, an essential splicing factor important for intron definition and alternative splicing. The cloning and characterization of Pompeii U2AF65 show it is highly similar to human U2AF65 in sequence and function and is more thermostable than the human protein when bound to RNA in vitro. Notably, Pompeii U2AF65 can restore splicing in a human extract depleted of human U2AF. We also determine that the general splicing mechanisms and signal sequences are conserved in the Pompeii worm, an annelid which has previously been uncharacterized in terms of splicing factors and signals.

Keywords: Splicing factor; U2AF65; Thermostable protein; Pre-mRNA splicing; Intron sequence

Promoter analyses of SCC antigen genes by Yoshinori Suminami; Fumio Kishi; Shugo Nawata; Akihiro Murakami; Yuko Sakaguchi; Kotaro Sueoka; Fumitaka Numa; Norihiro Sugino; Hiroshi Kato (pp. 208-212).

SCC antigen (SCCA) has been used as a tumor marker for squamous cell carcinoma. Analyses of the SCCA1 and SCCA2 genes, which are almost identical, and their promoters have been reported. Recently it was found that both SCCAs were stimulated by interleukin (IL)-4 and IL-13. Here we analyzed the promoter activity of both SCCAs in the 5′-flanking region, exon 1, and intron 1 to evaluate a putative STAT6 binding site. The addition of intron 1 to the luciferase assay constructs including the 5′-flanking region significantly augmented the promoter activity of both SCCA1 and SCCA2. Furthermore, deletion analyses of intron 1 revealed that a 50-bp fragment of intron 1 that includes putative STAT6 binding site was responsible for the increased promoter activity. Although the sequences of SCCA1 and SCCA2 are very similar in the 5′-flanking region, the analysis of the −337 single nucleotide polymorphism of SCCA2 indicated that this polymorphism may underlie the difference in promoter activity between SCCA1 and SCCA2.

Keywords: SCC antigen; SCC antigen 1; SCC antigen 2; Promoter; STAT6

Molecular cloning and characterization of calmodulin genes in young oak seedlings ( Quercus petraea L.) during early flooding stress by H�l�ne Folzer; Nicolas Capelli; James Dat; Pierre-Marie Badot (pp. 213-219).

As part of an integrated study on the molecular response of woody plants to flooding, three CaM genes were isolated from oak seedlings ( Quercus petraea Liebl.) and characterized. QpCaM-1 was almost exclusively expressed in roots, whereas QpCaM-2 and -3 were more evenly distributed throughout the plant. The present paper documents the differential expression of these genes during hypoxia.

Keywords: Abbreviations; aa; amino acid(s); CaM; calmodulin; RT-PCR; reverse transcription-polymerase chain reaction; UTR; untranslated region(s)Calmodulin; Flooding; Stress; Woody plant; Oak

Porcine SPLUNC1: Molecular cloning, characterization and expression analysis by Knud Larsen; Lone Bruhn Madsen; Christian Bendixen (pp. 220-226).

SPLUNC1, originally named PLUNC for palate, lung and nasal epithelium clone, is a small protein which is secreted from the epithelial cells of the nasal cavity and the upper respiratory tract in humans, mice, rats and cows. SPLUNC1 is structurally homologous to the two key mediators of host defense against Gram-negative bacteria, lipopolysaccharide binding protein (LBP) and bactericidal permeability increasing protein (BPI). SPLUNC1 is therefore believed to play a role in the innate immune system. This work reports the cloning and analysis of the porcine ( Sus scrofa) homologue of SPLUNC1. The SPLUNC1 cDNA was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotide primers derived from in silico sequences. The porcine cDNA codes for a protein of 249 amino acids which shows a high similarity to bovine (74%) and to human (69%) SPLUNC1. The predicted S. scrofa SPLUNC1, SsSPLUNC1, polypeptide contains a putative signal peptide of 19 residues. A similar signal sequence is also found in all other members of the PLUNC family. Expression analysis by RT-PCR demonstrated a very high expression level of the porcine SPLUNC1 homologue in trachea and lung tissue only. This airway-specific expression might be of particular interest in the study of airborne diseases in pig.

Keywords: Abbreviations; BPI; bactericidal permeability increasing protein; DIAS; Danish Institute of Agricultural Sciences; LBP; lipopolysaccharide binding protein; ORF; open reading frame; PCR; polymerase chain reaction; RT-PCR; reverse transcriptase PCR; UTR; untranslated regionAir passage; cDNA; Gene expression; Host defense; Lung; SPLUNC1; Sus scrofa

Gene Structure and Expression Author Index (pp. 227-228).

Gene Structure and Expression Cumulative Contents (pp. 229-230).

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BBA - Gene Structure and Expression (v.1727, #3)