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BBA - Gene Structure and Expression (v.1727, #1)
Does the AD7c-NTP locus encode a protein? by Jan Ole Kriegs; Jürgen Schmitz; Wojciech Makalowski; Jürgen Brosius (pp. 1-4).
AD7c-NTP, the only known protein entirely encoded by tandem and nested cassettes of Alu repetitive elements, is reportedly over-expressed in brains of Alzheimer's disease patients [de la Monte et al., J. Clin. Invest. 15 (1997)]. Based on these findings a commercial diagnostic assay (“7c Gold�/“AlzheimAlert� ) has been developed. We analyzed the published cDNA sequence and compared it to corresponding EST clones as well as the genomic sequences of human and chimpanzee. We come to the conclusion that the existence of the gene and in particular the predicted protein is inconsistent with EST and genomic data. Previously published data need to be reassessed.
Keywords: Alzheimer's disease; Neuronal thread protein; Alu element; Exaptation
Cloning and expression of levansucrase from Leuconostoc mesenteroides B-512 FMC in Escherichia coli by Hee Kyoung Kang; Mi Young Seo; Eun Seong Seo; Doman Kim; Seon Yong Chung; Atsuo Kimura; Donal F. Day; John F. Robyt (pp. 5-15).
Leuconostoc mesenteroides B-512 FMC produces dextran and levan using sucrose. Because of the industrial importance of dextrans and oligosaccharides synthesized by dextransucrase (one of glycansucrases from L. mesenteroides), much is known about the dextransucrase, including expression and regulation of gene. However, no detailed report about levansucrase, another industrially important glycansucrase from L. mesenteroides, and its gene was available. In this paper, we report the first-time isolation and molecular characterization of a L. mesenteroides levansucrase gene ( m1ft). The gene m1ft is composed of 1272-bp nucleotides and codes for a protein of 424 amino acid residues with calculated molecular mass of 47.1 kDa. The purified protein was estimated to be about 51.7 kDa including a His-tag based on SDS-PAGE. It showed an activity band at 103 kDa on a non-denaturing SDS-PAGE, indicating a dimeric form of the active M1FT. M1FT levan structure was confirmed by NMR and dot blot analysis with an anti-levan-antibody. M1FT converted 150 mM sucrose to levan (18%), 1-kestose (17%), nystose (11%) and 1,1,1-kestopentaose (7%) with the liberation of glucose. The M1FT enzyme produced erlose [ O-α-d-glucopyranosyl-(1→4)- O-α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside] as an acceptor product with maltose. The optimum temperature and pH of this enzyme for levan formation were 30 °C and pH 6.2, respectively. M1FT levansucrase activity was completely abolished by 1 mM Hg2+ or Ag2+. The Km and Vmax values for levansucrase were calculated to be 26.6 mM and 126.6 μmol min-1 mg-1.
Keywords: Leuconostoc mesenteroides; Levansucrase; Gene cloning
Identification and characterization of a family of Caenorhabditis elegans genes that is homologous to the cadmium-responsive gene cdr-1 by Jie Dong; Min Ok Song; Jonathan H. Freedman (pp. 16-26).
Six Caenorhabditis elegans genes that are homologous to the novel, cadmium-responsive gene cdr-1 have been identified and characterized. Nucleotide and amino acid sequence comparisons among the CDR family, which includes cdr-1, cdr-2, cdr-3, cdr-4, cdr-5, cdr-6, and cdr-7, reveals a high degree of identity among the seven members in this family. There are high levels of amino acid and nucleotide sequence similarity in the lengths of the open reading frames, predicted sizes, and protein characteristics. The seven proteins are predicted to be extremely hydrophobic, and are classified as integral membrane proteins. Structural analysis of the predicted proteins suggests that they may have similar biological functions. In response to cadmium exposure, cdr-1, cdr-2, cdr-3, and cdr-4 transcription significantly increases. In contrast, the levels of cdr-5, cdr-6, and cdr-7 transcription are not significantly affected or inhibited by cadmium exposure. Further, in non-exposed C. elegans, cdr-2, cdr-4, cdr-6, and cdr-7 are constitutively expressed. When CDR-1 expression was inhibited using RNAi, numerous fluid droplets were observed throughout the nematode body cavity. This phenotype became more pronounced in the presence of hypotonic stress. This suggests that CDR-1 may function in osmoregulation to maintain salt balance in C. elegans.
Keywords: Abbreviations; CDR; cadmium-responsive gene; RACE; rapid amplification of cDNA ends; RPA; RNase protection analysis; RNAi; RNA interference; MLC; myosin light chainCadmium; C. elegans; CDR-family; Membrane protein; Stress response; Osmoregulation; Transcription
Organization and functional analysis of the Schistosoma mansoni cathepsin D-like aspartic protease gene promoter by Alexandra Schulmeister; Oliver Heyers; Maria E. Morales; Paul J. Brindley; Richard Lucius; Grit Meusel; Bernd H. Kalinna (pp. 27-34).
We have cloned a 969-bp fragment of genomic DNA that spans 821 bp of the 5′ untranslated region, exon 1, a short intron, and part of exon 2 of the Schistosoma mansoni cathepsin D gene by inverse PCR. Inspection of this sequence revealed the presence of two TATA-box motifs, two inverted CCAAT-box (inverted NF-Y) motifs and sequences with homology to binding sites for the transcription factors, AP-1 and NF-Y. This sequence and deletion variants were cloned into reporter gene constructs, in order to examine the ability of these putative regulatory sequences to drive heterologous reporter gene activity. PCR products were cloned into the luciferase reporter vector pXP2. These reporter gene constructs were used to transform HeLa cells which were cultured and examined for luciferase activity. Additionally, HeLa cells transiently transfected with an EGFP reporter plasmid driven by the putative promoter from the S. mansoni cathepsin D gene were examined for EGFP transcripts and fluorescence. The 5′ untranslated region of the S. mansoni cathepsin D gene, from position −772 to +40 (translation start ATG), included functional regulatory sequences capable of driving luciferase and EGFP expression, whereas shorter fragments from position −264 or −185 to +40 were insufficient to drive reporter activities.
Keywords: Schistosoma mansoni; Aspartic protease promoter; Luciferase assay; HeLa cell transfection; Promoter analysis; Cathepsin D
Two tyrosine hydroxylase genes in teleosts by Judith Candy; Chris Collet (pp. 35-44).
We report the finding of two non-allelic genes encoding tyrosine hydroxylase (TH) from the diploid teleost barramundi Lates calcarifer. Barramundi TH1 is the homologue of the higher vertebrate TH genes and encodes a protein of 489 amino acids that shares 90% sequence identity to the THs of other teleost species. A second non-allelic tyrosine hydroxylase gene (TH2) encodes a protein of 472 amino acids and shares 62% identity with TH1 and the vertebrate THs. TH1 mRNA is found in the brain and kidney of barramundi while TH2 mRNA is found only in brain. The TH2 gene is also present in the genomes of the pufferfish Takifugu and zebrafish Danio. Estimates of the rates of nucleotide substitution suggest the teleost TH2 genes are selectively constrained although not to the degree seen in the TH1 genes. Differential regulation of the two TH genes is, however, indicated by differences in transcript distribution, the nature of the Ca2+-responsive elements found in the proximal promoter region and the lack of recognised phosphorylation sites in TH2. Preservation of two apparently functional TH genes in phylogenetically distant teleost species is consistent with the notion of partitioning of function between duplicate genes.
Keywords: Abbreviations; TH; tyrosine hydroxylase; PAH; phenylalanine hydroxylase; TPH; tryptophan hydroxylase; AAAH; aromatic amino acid hydroxylase; PCR; polymerase chain reaction; RT; reverse transcriptase; NTP; nucleotide triphosphate; SDS; sodium dodecyl sulfate; SSC; saline sodium citrate; UTR; untranslated region; IGF; insulin-like growth factor; CRE; cAMP-response element; K; a; rate of replacement site substitution; K; s; rate of synonymous site substitution; Mya; million years agoTyrosine hydroxylase; Teleost; Gene duplication
Transcriptional regulation of the human reduced folate carrier promoter C: synergistic transactivation by Sp1 and C/EBP β and identification of a downstream repressor by Scott G. Payton; Johnathan R. Whetstine; Yubin Ge; Larry H. Matherly (pp. 45-57).
The human reduced folate carrier (hRFC) is ubiquitously but differentially expressed in human tissues and its levels are regulated by up to six alternatively spliced non-coding regions (designated A1/A2, A, B, C, D, and E) and by at least four promoters. By transient transfections of HepG2 human hepatoma cells with 5′ and 3′ deletion constructs spanning 2883 bp of upstream sequence, a transcriptionally important region was localized to within 177 bp flanking the transcriptional start sites for exon C. By gel shift and chromatin immunoprecipitation assays, Sp1 and C/EBP β transcription factors were found to bind consensus elements (GC-box, CCAAT-box) within this region. The functional importance of these elements was confirmed by transient tranfections of HepG2 cells with hRFC-C reporter constructs in which these elements were mutated, and by co-transfections of Drosophila SL-2 cells with wild-type hRFC-C promoter and expression constructs for Sp1 and C/EBP β. Whereas both Sp1 and C/EBP β transactivated hRFC-C promoter activity, C/EBP α and γ were transcriptionally inert. Sp1 combined with C/EBP β resulted in a synergistic transactivation. In HepG2 cells, transfections with Sp1 and C/EBP β both increased endogenous levels of hRFC-C transcripts. By 3′ deletion analysis, a repressor sequence was localized to within 71 bp flanking the minimal promoter. On gel shifts, a novel transcriptional repressor was localized to within 30 bp. Collectively, these results identify transcriptionally important regions in the hRFC-C minimal promoter that include a GC-box and CCAAT-box, and suggest that cooperative interactions between Sp1 and C/EBP β are essential for hRFC-C transactivation. Another possible factor in the tissue-specific regulation of the hRFC-C region involves the downstream repressor flanking the minimal promoter.
Keywords: Abbreviations; RFC; reduced folate carrier; Mtx; methotrexate; hRFC; human RFC; ALL; acute lymphoblastic leukemia; 5′-UTRs; 5′-untranslated regions; bp; base pairs; MEM; Minimum essential medium Eagle; 5′-RACE; 5′-rapid amplification of cDNA ends; ChIP; chromatin immunoprecipitationReduced folate carrier; Methotrexate; Sp1; C/EBP; Transcription
Cloning and characterization of 5′ upstream promoter region of rat WAP gene by Jacek Jura; Jolanta Jura; Krzysztof Murzyn; Paulina Wegrzyn; Adrian Zarebski (pp. 58-64).
Regulatory regions of genes encoding milk proteins are frequently used to produce in the mammary gland of transgenic animals a variety of pharmaceutically and medically important human proteins. One such example is the whey acidic protein (WAP) promoter region, identified so far in the genome of mouse, rat, rabbit, camel, pig, brushtail possum and Tammar wallaby. The aim of the present study was cloning and characterization of the 5′ upstream promoter region of rat WAP gene. Using Genome Walking procedure, we cloned the region extending from −849 to −3671 bp. We have shown that there are two conserved regions highly similar to hypersensitive sites present in mouse and rabbit upstream region of WAP gene with binding sites for STAT5 transcription factor, essential for expression of WAP gene in mammary glands during lactation. We characterized dispersed and tandem repeats in the upstream region of rat WAP gen localized not far away from the translation initiation site.
Keywords: Whey acidic protein gene; STAT5; Rat; WAP; promoter; Mammary gland
Fish possess multiple copies of fgfrl1, the gene for a novel FGF receptor by Beat Trueb; Stephan C.F. Neuhauss; Stefan Baertschi; Thorsten Rieckmann; Christof Schild; Sara Taeschler (pp. 65-74).
FGFRL1 is a novel FGF receptor that lacks the intracellular tyrosine kinase domain. While mammals, including man and mouse, possess a single copy of the FGFRL1 gene, fish have at least two copies, fgfrl1a and fgfrl1b. In zebrafish, both genes are located on chromosome 14, separated by about 10 cM. The two genes show a similar expression pattern in several zebrafish tissues, although the expression of fgfrl1b appears to be weaker than that of fgfrl1a. A clear difference is observed in the ovary of Fugu rubripes, which expresses fgfrl1a but not fgfrl1b. It is therefore possible that subfunctionalization has played a role in maintaining the two fgfrl1 genes during the evolution of fish. In human beings, the FGFRL1 gene is located on chromosome 4, adjacent to the SPON2, CTBP1 and MEAEA genes. These genes are also found adjacent to the fgfrl1a gene of Fugu, suggesting that FGFRL1, SPON2, CTBP1 and MEAEA were preserved as a coherent block during the evolution of Fugu and man.
Keywords: Abbreviations; EST; expressed sequence tag; FGF; fibroblast growth factor; FGFRL; fibroblast growth factor receptor-like; LG; linkage group; PCR; polymerase chain reaction; UTR; untranslated region Danio rerio; Fibroblast growth factor receptor; FGFR; Fugu rubripes; Whole-genome duplication; Zebrafish
Molecular cloning and expression of cDNA coding for four spliced isoforms of casein kinase Iα in goldfish oocytes by Ryo Horiguchi; Mika Tokumoto; Yoshitaka Nagahama; Toshinobu Tokumoto (pp. 75-80).
Casein kinase I (CKI) is a member of the serine/threonine protein kinases and located in a separate group within the superfamily of eukaryotic protein kinases. CKI isoforms regulate several checkpoints of the cell cycle and meiosis. In higher eukaryotes, CKIα has four isoforms produced through the alternative splicing of two short inserts. Here, we report the cloning, sequencing and expression of four alternatively spliced isoforms of CKIα from goldfish ovary. The cloned cDNAs were 2099–3002-bp long and classified as CKIα, CKIαS, CKIαL and CKIαLS. It was revealed that two major (3.0 and 2.0 kb) messages were strongly expressed in the ovary. Four isoforms are expressed in previtellogenic to vitellogenic oocytes. In the huge nucleus of the oocyte, referred to as the germinal vesicle, CKIαS is dominant and CKIαL is expressed at a detectable level. Immunoblot analysis revealed that CKIα and CKIαS are major products in both immature and mature oocytes. These two isoforms were expressed in a tissue-dependent manner.
Keywords: Casein kinase Iα; Goldfish ovary; Isoform expression