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BBA - Gene Structure and Expression (v.1681, #2-3)
Composition and functional specificity of SWI2/SNF2 class chromatin remodeling complexes by Lisette Mohrmann; C. Peter Verrijzer (pp. 59-73).
By regulating the structure of chromatin, ATP-dependent chromatin remodeling complexes (remodelers) perform critical functions in the maintenance, transmission and expression of the eukaryotic genome. Although all known chromatin-remodeling complexes contain an ATPase as a central motor subunit, a number of distinct classes have been recognized. Recent studies have emphasized a more extensive functional diversification among closely related chromatin remodeling complexes than previously anticipated. Here, we discuss recent insights in the functional differences between two evolutionary conserved subclasses of SWI/SNF-related chromatin remodeling factors. One subfamily comprises yeast SWI/SNF, fly BAP and mammalian BAF, whereas the other subfamily includes yeast RSC, fly PBAP and mammalian PBAF. We review the subunit composition, conserved protein modules and biological functions of each of these subclasses of SWI/SNF remodelers. In particular, we will focus on the roles of specific subunits in developmental gene control and human diseases. Recent findings suggest that functional diversification among SWI/SNF complexes allows the eukaryotic cell to fine-tune and integrate the execution of diverse biological programs involving the expression, maintenance and duplication of its genome.
Keywords: SWI/SNF; Brahma; RSC; Chromatin remodeling; Cancer
Structural characterization of the gene encoding the large zinc finger protein ZAS3: implication to the origin of multiple promoters in eukaryotic genes by Joung-Woo Hong; Lai-Chu Wu (pp. 74-87).
ZAS3 is a large zinc finger protein that regulates κB-mediated transcription and TNF-driven signal transduction pathway. Herein, we have characterized the mouse ZAS3 gene that spans 400 kb and splits into 16 exons. Four ZAS3 exons, ranging from 676 to 3956 nucleotides, are significantly larger than the average size of mammalian internal exons. Intron 10, when retained in mRNAs, encodes N-terminal DNA binding domain, called ZASN. As predicted from cDNAs, 5′ untranslated region composed of the 2317 nucleotides is extremely long and contains upstream open reading frames, suggesting that translation initiation of ZAS3 transcripts by conventional cap-dependent ribosome scanning mechanism may be inefficient. Additionally, cDNA data analysis followed by reporter gene assays shows that the ZAS3 locus harbors two promoters that are 80 kb apart. The data suggest that the expression of ZAS3 is controlled by a combination of differential promoter usage, alternative splicing, and possible intergenic splicing. The distribution and degree of conservation of exons within the ZAS3 locus, together with the complex alternative splicing events and upstream open reading frame in 5′ untranslated exons, lead us to speculate that multiple promoters of an eukaryotic gene might be residual traces of regulatory regions of other genes lost in evolution.
Keywords: ZAS3; Multiple promoter; Gene within gene; Intergenic splicing; Separated C; 2; H; 2; zinc finger pair; Transcriptional interference
Functional analysis of the mouse Fcgrt 5′ proximal promoter by Bhavna Tiwari; Richard P. Junghans (pp. 88-98).
The Brambell receptor (FcRB, FcRn) mediates transmission of immunity from mother to young perinatally and plays a central role in IgG protection and homeostasis throughout life. Developmental and tissue specific expression is via transcriptional regulation of the gene for the receptor heavy chain, Fcgrt. In neonatal mouse intestine, expression is high to absorb IgG from mother's milk, but then nearly absent in adult intestine and in most other tissues. In this initial functional characterization, reporter gene assays revealed at least two upstream promoter regions (−372/−140 and −105/−1), each with repressor and activator functions, and a downstream activator domain (+1/+78). The gene carries no upstream TATA element or Inr sequence, and an apparent downstream promoter feature (DPE) lacks typical context for an active element, rendering uncertain the nature of the organizing feature for the transcription initiation complex. Electrophoretic mobility shift analysis of the proximal upstream region identified transcription factor (TF) binding to motifs for NF1, Sp1 (GT box) and Ets. Binding to the GT box and Ets motif was observed only with mature cell sources, and not with neonatal enterocyte extracts. Site-directed mutagenesis confirmed that TF binding to the GT box up-regulates promoter activity in adult (NIH3T3) cells, whereas binding to the Ets motif represses activity. Binding of NF1 protein to the Fcgrt promoter was confirmed in nuclear extracts of NIH3T3 cells and in adult mouse enterocytes, whereas an apparently different TF bound uniquely to the NF1 site in neonatal enterocyte extracts as the sole identified candidate for an expected developmental-specific high-level activator in this tissue. These data indicate regions of potential importance in the Fcgrt proximal promoter and additionally suggest that the selective temporal and spatial availability of specific TFs may contribute to the developmental and tissue-specific regulation of Fcgrt expression.
Keywords: Brambell receptor; FcRB; FcRp; FcRn; IgG receptor; Transcription; Gene regulation
Comparative expression analysis of two paralogous Hsp70s in rainbow trout cells exposed to heat stress by Nobuhiko Ojima; Michiaki Yamashita; Shugo Watabe (pp. 99-106).
Heat-shock protein 70 (Hsp70) is the major stress-inducible protein in vertebrates and highly conserved throughout evolution. To accurately investigate the mRNA expression profiles of multiple Hsp70s in rainbow trout Oncorhynchus mykiss, we isolated full-length cDNA clones encoding Hsp70 from the fish and investigated their mRNA expression profiles during heat stress. Consequently, two Hsp70s, Hsp70a and Hsp70b, were identified and found to have 98.1% identity in their deduced amino acid sequences. Southern blot analysis indicated that the two Hsp70s are encoded by distinct genes in the genome. Northern blot analysis showed that each of Hsp70a and Hsp70b expressed two mRNA species having different sizes by heat stress in rainbow trout RTG-2 cells. The induction levels of total Hsp70b mRNAs were consistently higher than Hsp70a counterparts during heat stress, although the expression profiles of the two genes were similar to each other in temperature shift and time course experiments. Interestingly, an mRNA species with a larger molecular size was expressed only under severe heat stress not less than 28 °C irrespective of Hsp70a and Hsp70b. These results suggest that the comprehensive identification of duplicated genes is a prerequisite to examining the gene expression profiles for tetraploid species such as rainbow trout.
Keywords: Gene expression; Heat shock; Hsp70; Northern blotting; Rainbow trout
The ornithine cycle enzyme arginase from Agaricus bisporus and its role in urea accumulation in fruit bodies by Matthijs J.M. Wagemaker; Willem Welboren; Chris van der Drift; Mike S.M. Jetten; Leo J.L.D. Van Griensven; Huub J.M. Op den Camp (pp. 107-115).
An extensive survey of higher fungi revealed that members of the family Agaricaceae, including Agaricus bisporus, accumulate substantial amounts of urea in their fruit bodies. An important role of the ornithine cycle enzymes in urea accumulation has been proposed. In this work, we present the cloning and sequencing of the arginase gene and its promoter region from A. bisporus. A PCR-probe based on fungal arginase was used to identify the A. bisporus arginase gene from a cDNA library. The arginase cDNA encodes a 311-aa protein which is most likely expressed in the cytosol. Expression of the cDNA in Escherichia coli was established as a His-tagged fusion protein. The arginase gene was used as a molecular marker to study expression and regulation during sporophore formation and postharvest development. The expression of the arginase gene was significantly up-regulated from developmental stage 3 onwards for all the tissues studied. A maximum of expression was reached at stage 6 for both stipe and cap tissue. In postharvest stages 5, 6 and 7 the level of expression observed was similar to normal growth stages 5, 6 and 7. A good correlation was found between arginase expression and urea content of stipe, velum, gills, cap and peel tissue. For all tissues the urea content decreased over the first four stages of development. From stage 4 onwards urea accumulated again except for stipe tissue where no significant changes were observed. The same trend was also observed for postharvest development, but the observed increase of urea in postharvest tissues was much higher.
Keywords: Urea-cycle; Nitrogen metabolism; Basidiomycete; Post harvest; Fungus; Mushroom
Molecular characterization of the glutamine synthetase gene in the Pacific oyster Crassostrea gigas: expression study in response to xenobiotic exposure and developmental stage by Arnaud Tanguy; Isabelle Boutet; Dario Moraga (pp. 116-125).
In this study, we characterized the full-length cDNA and genomic sequence of the gene encoding cytosolic glutamine synthetase ( CgGSII) in the Pacific oyster, Crassostrea gigas. A phylogenetic analysis of GS sequences showed that CgGS clustered with the invertebrate group as expected. We analyzed the expression of mRNA CgGSII using RT-PCR to follow the expression of this gene in gills and digestive gland of oysters exposed, under experimental conditions, to hypoxia and to several contaminants (hydrocarbons and two pesticide treatments, glyphosate and a mixture of atrazine, diuron and isoproturon). We also investigated the expression of CgGSII in different developmental stages of C. gigas. Our results show that CgGSII expression was highly regulated in xenobiotic-exposed oysters compared to the control for all the treatments. Likewise, CgGSII expression was highly regulated according to the developmental stage of C. gigas. Finally, use of CgGSII as a possible marker to monitor xenobiotic exposure in disturbed ecosystems is discussed.
Keywords: Mollusc; Pesticide; Hypoxia; Hydrocarbon; Larva
The p100 EBNA-2 coactivator: a highly conserved protein found in a range of exocrine and endocrine cells and tissues in cattle by Marita K. Broadhurst; Rita S.-F. Lee; Sarah Hawkins; Thomas T. Wheeler (pp. 126-133).
The p100 transcriptional coactivator is an evolutionarily conserved protein that has been shown to be a coactivator of the Epstein–Barr virus-encoded transcription factor EBNA-2, as well as Stat5 and Stat6. However, the p100 genomic organisation, phylogeny and expression have not been analysed in detail and its physiological role is uncertain. The cDNA and amino acid sequence of bovine p100 was obtained, and the genomic organisation of the human p100 gene was determined. Homologues of p100 were found in the genomes of 21 diverse eukaryotes. Western blot and immunohistochemical analyses revealed that the bovine p100 protein is present in a range of exocrine and endocrine cells and tissues, including the lactating mammary gland, pancreas, adrenal, parotid, anterior pituitary, corpus luteum, ovarian follicular cells, placenta and small intestine. P100 was present in the nuclei of mammary epithelial cells and pancreatic acinar cells, but only in the extranuclear compartment of the other immunopositive tissues. These data indicate that the p100 protein plays a fundamental role in eukaryotic biology, and functions in secretory cells, at least in cattle.
Keywords: Abbreviations; nt; nucleotide; aa; amino acid; UTR; untranslated region; PCR; polymerase chain reaction; RACE; rapid amplification of cDNA ends; RT-PCR; reverse transcription polymerase chain reaction; IPTG; isopropyl β-; d; -1-thiogalactopyranoside; ORF; open reading frame; EBNA-2; Epstein–Barr virus nuclear antigen-2; 4SNc; four staphylococcal nuclease homology domains; TFIIE; transcription factor II E; cDNA; DNA complementary to RNA; BLAST; basic logical alignment of sequences tool; PSI-BLAST; position-specific iterated BLASTTudor domain; Nuclease; Mammary; Pancreas; 4SNc-tudor
Molecular characterization of NADase-streptolysin O operon of hemolytic streptococci by Hisashi Kimoto; Yutaka Fujii; Yoshifumi Yokota; Akira Taketo (pp. 134-149).
Whether slo, the gene encoding streptolysin O (SLO), a streptococcal cytolysin, has its own promoter or not is unsettled as yet. Present analyses demonstrate that slo is a member of an operon covering the upper-stream nusG and nga (NADase) genes, from which transcription of slo proceeds polycistronically, and major transcript is produced by readthrough from nga promoter. Mutational conversion of the sixth nucleotide T at the putative −10 region of chromosomal nga gene into C caused a drastic decrease in both NADase and SLO activities and the disappearance of the two corresponding mRNA bands from the Northern blot profile. The initiation site of the transcription was determined at 56 bp upstream (NusG gene) and 25 bp upstream (NADase gene) of each initiation codon. Although the promoter region of slo gene is highly conserved between group A and C streptococci, the proper slo promoter is nonfunctional in group C strain H46A. Moreover, commonly conserved arrangement was limited to the nusG-nga-orf1-slo region. These results indicate an intimate relationship between NADase and SLO in the regulation of their biosynthesis. Additional results suggest that NADase, synthesized as precursor with feeble activity, is activated by removing the carboxyl terminal region during or after secretion into culture medium.
Keywords: Abbreviations; Ap; ampicillin; Em; erythromycin; Sp; spectinomycin; Tet; tetracycline; THY medium; Todd Hewitt broth supplemented with 1% yeast extract; LB; Luria-Bertani broth; BSA; bovine serum albumin; EDTA; ethylene diamine tetraacetic acid; TE; tris–EDTA buffer; SSC; standard saline citrate; PBST; phosphate-buffered saline containing 0.05% Tween 20; SDS; sodium dodecylsulfate; PAGE; polyacrylamide gel electrophoresis; CBB; Coomassie brilliant blue; PVDF; polyvinylidene difluoride; PCR; polymerase chain reaction; RT; reverse transcription; dGTP; deoxyguanosine 5′-triphosphate; TS; thermosensitive; ori; replication origin; ORF; open reading frame; HU; Hemolytic activity; SLO; streptolysin O; SLS; streptolysin S; ASLO; anti-streptolysin O serum; NADase; NAD-glycohydrolase; nga; NADase gene; sag; SLS-associated gene; CFU; colony-forming units; GAS; group A; Streptococcus; GBS; group B; Streptococcus; GCS; group C; Streptococcus; GGS; group G; Streptococcus; bp; base pairStreptococci; Gene targeting; Streptolysin; SLO; NADase; nga
Alternatively spliced variants of protocadherin 8 exhibit distinct patterns of expression during mouse development by Helen Makarenkova; Hiroko Sugiura; Kanato Yamagata; Geoffrey Owens (pp. 150-156).
Protocadherins, a subgroup of the cadherin superfamily of calcium-dependent cell adhesion molecules, are considered to play important roles in the developing embryo particularly in the central nervous system. The Protocadherin 8 ( Pcdh8) gene comprises three coding exons in both human and mouse, and the exon junctions are precisely conserved between these two species. Alternative splicing of Pcdh8 RNA leads to the formation of two isoforms that differ in the length of the cytoplasmic domains. We have investigated the expression of these short and long variants of Pcdh8 during early mouse development by RT/PCR and in situ hybridization. We found that both isoforms were predominantly expressed in the nervous system, and that their expression patterns appeared to be developmentally regulated. However, the short variant had a broader pattern of expression than the long variant and was found in some non-neuronal tissues, such as paraxial mesoderm, developing somites, and in limb interdigital mesenchyme where massive programmed cell death occurs. The differential expression of two alternative cytoplasmic domain variants suggests that Pcdh8 may regulate cell adhesion in a variety of developmental processes, and that this may involve different intracellular interactions.
Keywords: Protocadherin; Cell adhesion; Alternative RNA splicing; Mouse development; Nervous system
Identification and characterization of hepsin/-TM, a non-transmembrane hepsin isoform by Yang Li; Zhenbao Yu; Xin Zhao; Shi-Hsiang Shen (pp. 157-165).
Type II transmembrane serine proteases (TTSPs), including hepsin, are a new class of cell surface catalytic enzymes. In the present study, a non-transmembrane isoform of hepsin, named hepsin/-TM that originates from alternative splicing, was identified. Unlike the transmembrane hepsin isoform, this non-transmembrane isoform was distributed within the cytoplasm. Real-time PCR experiments revealed that while hepsin was expressed in all tested human tissues, hepsin/-TM was restricted in kidney, brain and lung tissues. Significantly, hepsin/-TM was not expressed in liver where hepsin was originally identified. However, hepsin/-TM was highly expressed in brain where hepsin was expressed at a relatively lower level. Moreover, these two isoforms showed different expression patterns in a number of colon adenocarcinoma cell lines. In addition, in contrast to hepsin, expression of hepsin/-TM in vivo does not exert any apparent inhibitory effect on mammalian cell growth.
Keywords: Type II transmembrane serine protease; Hepsin; Isoform; Alternative splicing; Expression pattern
Translational control by internal ribosome entry site in Saccharomyces cerevisiae by Ayako Seino; Yasuko Yanagida; Masuo Aizawa; Eiry Kobatake (pp. 166-174).
To confirm the active involvement of the internal ribosome entry site (IRES)-dependent translation in living eukaryotes, Saccharomyces cerevisiae and HAP4 IRES were used for in vitro and in vivo experiments. Since HAP4 protein might be required for activating mRNAs transcription in yeast cells when they are released from catabolite repression, the translational efficiency of HAP4 mRNA is presumed to increase under such a condition. The in vitro experiment showed clearly that the translational mechanism was shifted from the cap-dependent to the completely IRES-dependent translation when the yeast cells were derepressed from catabolite repression. From in vivo experiment, it was confirmed that the IRES-dependent translational efficiency was in a low level at the beginning of the stationary growth phase, and was enhanced at the glucose-exhausted phase. These results indicate that yeast cells on the catabolite derepressed condition could get a large amount of HAP4 protein for the completely IRES-dependent translation, while the IRES-dependent translational efficiency is increased. It has been proven that IRES functions directing the initiation of translation in living yeast cells.
Keywords: IRES; Saccharomyces cerevisiae; HAP4; Cap-independent translation
Transcriptional regulation of the mouse microtubule-associated protein tau by Lei Gao; Kerry L. Tucker; Athena Andreadis (pp. 175-181).
The microtubule-associated protein (MAP) tau is found primarily in neurons and errors in its regulation are associated with Alzheimer's disease and other neurodegenerative disorders. Tau expression is transcriptionally regulated and tissue-specific. In this study, starting with a ∼7500-bp fragment from the mouse tau gene, which includes tau exon −1, we define regions preferentially conferring tissue-specific expression. Furthermore, gel shift assays indicate that transcriptional regulators SP-1 and AP-2 are important for basal expression but not necessary for neuron-specific expression of the tau transcript.
Keywords: Abbreviations; bp; base pairs; CAT; chloramphenicol acetyltransferase; kb(p); kilobase (pairs); MAP; microtubule-associated protein; MTP; mouse tau promoter; NFTs; neurofibrillary tangles; nt; nucleotidesMouse tau; Promoter specificity; Transcriptional regulation; Factors AP-2, SP-1