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BBA - Gene Structure and Expression (v.1728, #3)

Editorial Board (pp. ii).

Two novel arginine/serine (SR) proteins in maize are differentially spliced and utilize non-canonical splice sites by Smriti Gupta; Bing-Bing Wang; Gabrielle A. Stryker; Mar�a Eugenia Zanetti; Shailesh K. Lal (pp. 105-114).

The serine–arginine (SR)-rich splicing proteins are highly conserved RNA binding nuclear phosphor-proteins that play important roles in both regular and alternative splicing. Here we describe two novel putative SR genes from maize, designated zmRSp31A and zmRSp31B. Both genes contain characteristic RNA binding motifs RNP-1 and RNP-2, a serine/arginine-rich (RS) domain and share significant sequence similarity to the Arabidopsis atRSp31 family of SR proteins. Both zmRSp31A and zmRSp31B produce multiple transcripts by alternative splicing, of which majority of the alternatively spliced transcripts utilize non-canonical splice sites. zmRSp31A and zmRSp31B produce at least six and four transcripts, respectively, of which only one corresponds to the wild type proteins for each gene. All the alternatively spliced transcripts of both the genes, with one exception, are predicted to encode small truncated proteins containing only the RNP-2 domain of their first RNA recognition motif and completely lack the carboxyl terminal RS domain. We provide evidence that some of the alternatively spliced transcripts of both genes are associated with polysomes and interact with the translational machinery.

Keywords: Alternative splicing; Pre-mRNA splicing; EST analysis; Non-canonical splice sites

Characterization of the basal promoter element of the human type 5 17beta-hydroxysteroid dehydrogenase gene by Kenan Qin; Robert L. Rosenfield (pp. 115-125).

Testosterone biosynthesis from androstenedione is carried out by androgenic 17β-hydroxysteroid dehydrogenase (17β-HSD) activity. Of the androgenic 17β-HSD isoenzymes, only type 5 (17β-HSD5) is expressed ubiquitously, including the human adrenal gland and ovary. To characterize this gene promoter, luciferase constructs of the human 5′-flanking region were transiently transfected into the H295R human adrenal carcinoma cell line. A series of fragment deletion constructs and electrophoretic mobility shift assays suggested that a sequence of CCTCCTCCT at −65 to −58 bp is the core sequence and demonstrated Sp1/Sp3 binding to this CCT repeat. Forskolin stimulated the promoter activity of the HSD17B5 gene through this Sp1/Sp3 binding site. Mutation of this site resulted in a significant reduction of HSD17B5 promoter basal and forskolin-induced activity. Mithramycin A, which inhibits the binding of Sp1 and Sp3 to DNA, also remarkably decreased HSD17B5 mRNA expression in the H295R cell line. These results indicate that members of the Sp family of transcription factors play an important role in regulating constitutive and stimulated expression of the HSD17B5 gene in H295R cells.

Keywords: Abbreviations; AKR; aldo-keto reductase; AP2; activator protein-2; EMSA; electrophoretic mobility shift assay; Egr-1; early growth response factor 1; Ets-1; E26 transformation specific-1; HSD; hydroxysteroid dehydrogenase; PCOS; polycystic ovary syndrome; PCR; polymerase chain reaction; RT-PCR; reverse transcriptase-polymerase chain reaction; Sp1/3; stimulatory protein 1/3Type 5 17beta-hydroxysteroid dehydrogenase gene (HSD17B5); Sp1/Sp3; Promoter regulation

Model of transcriptional regulation of the BRCA1–NBR2 bi-directional transcriptional unit by Ting-Chung Suen; Moon-shong Tang; Paul E. Goss (pp. 126-134).

In contrast to hundreds of mutations found in familial breast and/or ovarian cancers, somatic mutations of BRCA1 are very rare. However, a high percentage of sporadic breast and ovarian cancers show a reduction in BRCA1 expression, suggesting that defects in transcriptional regulation is a contributing factor. BRCA1 shares a promoter with its neighboring gene, NBR2, which is transcribed in the opposite direction. We have previously shown that the transcription of BRCA1 is negatively regulated by protein factors that interact with a 36-bp segment, located 575 bp into its first intron. We now report the localization of an 18-bp transcriptional repressor element for NBR2, which resides 948 bp into its first intron. The binding of nuclear proteins to this repressor element was detected by electrophoretic mobility shift assays (EMSAs), and it conferred an orientation-dependent functional suppression onto a heterologous thymidine kinase promoter. Combined with our previous studies, a model of transcriptional regulation of the closely aligned BRCA1–NBR2 bi-directional unit is proposed. A minimal 56-bp DNA region is functional in driving transcription in both directions, while uni-directional control is provided by distinct repressors that bind to sequences located in the first intron of the respective genes.

Keywords: BRCA1; NBR2; Transcription; Transcriptional repressor; Tumor suppressor gene

Translational effects of differential codon usage among intragenic domains of new genes in Drosophila by Jianming Zhang; Manyuan Long; Liming Li (pp. 135-142).

Evolved codon usages often pose a technical challenge over the expressing of eukaryotic genes in microbial systems because of changed translational machinery. In the present study, we investigated the translational effects of intragenic differential codon usage on the expression of the new Drosophila gene, jingwei ( jgw), a chimera derived from two unrelated parental genes: Ymp and Adh. We found that jgw possesses a strong intragenic differential usage of synonymous codons, i.e. the Adh-derived C-domain has a significantly higher codon bias than that of the Ymp-derived N-domain ( P=0.0023 by t-test). Additional evolutionary analysis revealed the heterogeneous distribution of rare codons, implicating its role in gene regulation and protein translation. The in vitro expression of jgw further demonstrated that the heterogeneous distribution of rare codons has played a role in regulating gene expression, particularly, affecting the quality of protein translation.

Keywords: Abbreviations; Adh; alcohol dehydrogenase; jgw; jingwei; gene; ENC; effective number of codonsCodon usage; Intragenic codon bias; Evolution of new genes; Analysis of protein functions; Expression of insect genes; Short-chain dehydrogenase

Bacillus subtilis tRNA^Pro with the anticodon mo⁵UGG can recognize the codon CCC by Yuko Yamada; Jitsuhiro Matsugi; Hisayuki Ishikura; Katsutoshi Murao (pp. 143-149).

In Bacillus subtilis, four codons, CCU, CCC, CCA, and CCG, are used for proline. There exists, however, only one proline-specific tRNA having the anticodon mo⁵UGG. Here, we found that this tRNA^Pro(mo⁵UGG) can read not only the codons CCA, CCG and CCU but also CCC, using an in vitro assay system. This means that the first nucleoside of its anticodon, 5-methoxyuridine (mo⁵U), recognizes A, G, U and C. On the other hand, it was reported that mo⁵U at the first position of the anticodon of tRNA^Val(mo⁵UAC) can recognize A, G, and U but not C. A comparison of the structure of the anticodon stem and loop of tRNA^Pro(mo⁵UGG) with those of other tRNAs containing mo⁵U at the first positions of the anticodons suggests that a modification of nucleoside 32 to pseudouridine (Ψ) enables tRNA^Pro(mo⁵UGG) to read the CCC codon.

Keywords: tRNA; ^Pro; Codon specificity; Modified nucleoside; Bacillus subtilis

Transgene copy number-dependent rescue of murine β-globin knockout mice carrying a 183 kb human β-globin BAC genomic fragment by Jim Vadolas; Hady Wardan; Marco Bosmans; Faten Zaibak; Duangporn Jamsai; Lucille Voullaire; Robert Williamson; Panos A. Ioannou (pp. 150-162).

We report the generation and characterisation of the first transgenic mice exclusively expressing normal human β-globin (^huβ-globin) from a 183 kb genomic fragment. Four independent lines were generated, each containing 2–6 copies of the^huβ-globin locus at a single integration site. Steady state levels of^huβ-globin protein were dependent on transgene copy number, but independent of the site of integration. Hemizygosity for the transgene on a heterozygous knockout background (^huβ^+/0,^muβ^th-3/+) complemented fully the hematological abnormalities associated with the heterozygous knockout mutation in all four lines. Importantly, the rescue of the embryonic lethal phenotype that is characteristic of homozygosity for the knockout mutation was also demonstrated in two transgenic lines that were homozygous for two copies of the^huβ-globin locus, and in one transgenic line, which was hemizygous for six copies of the^huβ-globin locus. Our results illustrate the importance of transgene copy number determination and of the hemizygosity/homozygosity status in phenotypic complementation studies of transgenic mice containing large heterologous transgenes. Transgenic mouse colonies with 100%^huβ-globin production from the intact^huβ-globin locus have been established and will be invaluable in comparative and gene therapy studies with mouse models containing specific β-thalassemia mutations in the^huβ-globin locus.

Keywords: Bacterial artificial chromosome; β-Globin locus; Transgenic mice; Knockout mice; Transgene complementation

Differentially expressed nucleolar TGF-β1 target (DENTT) shows tissue-specific nuclear and cytoplasmic localization and increases TGF-β1-responsive transcription in primates by Laurent L. Ozbun; Alfredo Mart�nez; Sonia B. Jakowlew (pp. 163-180).

Differentially Expressed Nucleolar TGF-β1 Target (DENTT) is a new member of the TSPY/TSPY-like/SET/NAP-1 (TTSN) superfamily whose mRNA is induced by TGF-β1 in TGF-β1-responsive human lung cancer cells. Monkey DENTT mRNA contains a 2085-bp open reading frame that encodes a predicted polypeptide of 695 amino acids with five nuclear localization signals, two coiled-coil regions, and a domain that shows significant identity to a region that defines the TTSN superfamily. RT-PCR amplification and Western blot analyses showed DENTT mRNA and protein in adult monkey tissues, including the adrenal gland, cerebral cortex, and ovary. Immunohistochemical staining showed that numerous neurons were intensely immunoreactive for DENTT, as were anterior pituitary secretory cells, thyroid follicular cells, and smooth muscle cells of arteries and lung bronchial walls. DENTT expression was also prominent in monkey bronchiolar-alveolar adenomas and cell lines. While the addition of TGF-β1 or retinoic acid to monkey normal lung bronchial 12MBr6 cells and human lung cancer NCI-H727 cells increased DENTT protein production, TGF-β1 together with retinoic acid resulted in a more sustained increase in DENTT production than with TGF-β1 or retinoic acid alone. Transient transfection studies showed that ectopic DENTT expression significantly increased TGF-β1-responsive 3TP-Lux and CAGA12-Lux reporter transcription in 12MBr6 and NCI-H727 cells with TGF-β1 addition, while ectopic DENTT expression had no significant effect on the transcription of a retinoic acid-responsive element reporter in the presence of retinoic acid or TGF-β1. These findings suggest new possibilities for DENTT as a TGF-β1-regulated, but not a retinoic acid-regulated member of the TTSN superfamily in primate physiology.

Keywords: Differentially expressed nucleolar TGF-β1 target; TSPY/TSPY-like/SET/NAP-1; Monkey; Immunohistochemistry; Nucleus; Transcription

An Ets element regulates the transcription of the human 2B4 gene in natural killer cells by Swapnil V. Vaidya; Porunelloor A. Mathew (pp. 181-185).

2B4 (CD244) acts as an activation receptor on human NK cells, whereas it sends inhibitory signals in murine NK cells. A previous study indicated a prominent role for AP-1 in the transcription of 2B4 gene. To further understand the transcriptional regulation we analyzed the upstream positive regulatory region (−1151 to −704) of the 2B4 promoter. We have identified an Ets element that regulates the 2B4 gene transcription in an AP1 dependent manner.

Keywords: 2B4 promoter; Transcription regulation; Natural killer cell

Isolation, sequencing, and functional analysis of the TATA-less human ATPase II promoter by Tomasz Sobocki; Farah Jayman; Malgorzata B. Sobocka; Ruth Duchatellier; Probal Banerjee (pp. 186-198).

Multiple lines of evidence indicate that the P-type Mg²⁺-ATPase, termed ATPase II, could play an important role in apoptosis. With the long-term objective of studying the regulation of this protein during apoptosis, we delineated the exon–intron organization of the human ATPase II gene (within chromosome 4). Subsequently, we used RNA ligase-mediated rapid amplification of cDNA ends to identify a major transcription start site at position −143 with respect to the translation start site. Luciferase reporter analysis of a 1.2-kb 5′-flanking sequence (−1222 to +94 with respect to the transcription start site) revealed strong promoter activity in three human cell lines, human oligodendroglioma (HOG), SHSY5Y (hybrid neuroblastoma), and EA.hy926 (endothelial cell line). Serial deletions from the 5′ end of this sequence up to nucleotide −291 yielded some decrease in activity only in the EA.hy926 cells. Further deletion to −217 caused a drastic decrease in activity in all three cell lines, but a −148 fragment showed preferential reduction in activity in the EA.hy926 cells. The promoter activity was nearly equal in two sequence variants of the promoter, one of which (designated as Variant 2) contained a 15-bp direct repeat within a GC-rich region. Additionally, there were several single base-pair changes from the sequence reported by the human genome project. Despite the presence of enhancer/repressor elements, such as Sp1 and NFκB, relatively small differences in promoter activity were observed in the three cell lines. However, it is likely that such sequence elements could cause major regulation of promoter activity in cells subjected to conditions that trigger apoptosis. The ATPase II promoter sequence will provide valuable clues to the regulation and role of the ATPase II protein.

Keywords: Abbreviations; HGP; Human Genome Project; TF; transcription factor; RLM-RACE; RNA ligase-mediated rapid amplification of cDNA ends; APTL; aminophospholipid translocaseP-type ATPase; Promoter; Human ATPase II; Apoptosis; Aminophospholipid translocase

Isolation and characterization of a novel PHGPx gene in Raphanus sativus by Xiao-Dong Yang; Wen-Jun Li; Jin-Yuan Liu (pp. 199-205).

A full-length cDNA encoding putative phospholipid hydroperoxide glutathione peroxidase (PHGPx) was cloned from Raphanus sativus. The cDNA, designated RsPHGPx, includes an open reading frame which encodes 197 amino acid residues. The alignment of amino acid sequences showed that RsPHGPx had the highest sequence homology to plant PHGPx and contained an N-terminal extension characteristic of a mitochondrial targeting peptide. Northern blot analysis indicated that RsPHGPx was constitutively and ubiquitously expressed during radish development, and its expression was differently regulated by various stress conditions. The expression of RsPHGPx in a yeast PHGPx-deletion mutant significantly rescued the mutant sensitivity to oxidation-sensitive linolenic acid, just as the yeast PHGPx3 gene did. This suggested that RsPHGPx encodes a functional PHGPx protein.

Keywords: Abbreviations; GPx; glutathione peroxidase; PHGPx; phospholipid hydroperoxide glutathione peroxidase; t-BHP; tert; -butyl hydroperoxide; ORF; open reading frame Raphanus sativus; Phospholipid hydroperoxide glutathione peroxidase; Expression pattern; Oxidative stress; Yeast complementation

Gene structure and expression cumulative contents (pp. 207-208).

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BBA - Gene Structure and Expression (v.1728, #3)