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BBA - Gene Structure and Expression (v.1731, #3)

Editorial Board (pp. ii).

Molecular characterization of the insulin-like growth factor-I ( IGF-I) gene in channel catfish ( Ictalurus punctatus) by LaTonya A. Clay; Shiao Y. Wang; William R. Wolters; Brian C. Peterson; Geoffrey C. Waldbieser (pp. 139-148).

The insulin-like growth factor I (IGF-I) gene was characterized in channel catfish. Partial cDNA sequence, missing exon 1 and part of exon 2, was obtained in 5′- and 3′-RACE experiments. Direct sequencing of two bacterial artificial chromosome clones revealed gene structure and provided sequence from 640 bp upstream of the initiator methionine to 136 bp beyond the polyadenylation site. Genomic sequence contained a putative TATA box 506 bp upstream of the initiator methionine. The 477-bp reading frame within five exons encoded a 159-amino acid (aa) pre-propeptide highly similar to IGF-I in higher vertebrates. The sequence encoding the signal peptide was unique in catfish and contained 70% G+C content with the potential for a stable stem–loop structure. Full-length cDNA was only maintained in recombination-deficient (DH10B) strain E. coli. Levels of IGF-I mRNA were highest in liver, followed by brain and muscle, then heart and kidney ( P<0.05). A CT/GA dinucleotide microsatellite in intron 1 was highly polymorphic in commercial channel catfish, and permitted placement of the IGF-I gene on the catfish genetic map. However, specific IGF-I alleles were not correlated with differences in growth rate from 100 to 130 days post-hatch in USDA103 line catfish.

Keywords: Development; Hormone; Growth; Microsatellite; Linkage

Identification and characterization of Spinocerebellar Ataxia Type 7 (SCA7) isoform SCA7b in mice by Anna-Lena Str�m; Lars Forsgren; Monica Holmberg (pp. 149-153).

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease primarily affecting the cerebellum, brainstem and retina. The disease is caused by a polyglutamine expansion in ataxin-7, a protein with largely unknown function. To improve our knowledge of the expression and function of wild-type and expanded ataxin-7, we looked for alternative SCA7 transcripts in mice. We identified a murine SCA7 isoform (SCA7b) containing an uncharacterized exon homologous to the newly identified human exon 12b. Northern blot analysis revealed three exon 12b containing transcripts with molecular sizes of 7.5, 4.4 and 3.0 kb in mice. This contrasted with the situation in humans, where only one exon 12b-containing transcript was observed. Furthermore, Northern blot of the human 4.4 kb SCA7b isoform predominantly showed expression in the brain, while expression of both the murine 7.5-kb and the 4.4-kb transcripts were observed in several tissues including brain, heart, liver, kidney and testis. Quantitative real-time RT-PCR analysis revealed that in muscle and heart SCA7b is the predominant SCA7 isoform, while in brain equal levels of SCA7a and SCA7b was observed. Insertion of exon 12b into the murine SCA7 ORF resulted in a frame-shift that gave rise to an alternative ataxin-7 protein (ataxin-7b). The novel 58-amino acid C-terminus in ataxin-7b directed the protein to a more cytoplasmic location.

Keywords: Polyglutamine; ataxin-7; CAG repeat

ISL1 physically interacts with BETA2 to promote insulin gene transcriptional synergy in non-β cells by Si-Yang Peng; Wei-Ping Wang; Jun Meng; Tao Li; Hui Zhang; Yan-ming Li; Ping Chen; Kang-Tao Ma; Chun-Yan Zhou (pp. 154-159).

ISL1 is a LIM homeodomain protein that plays an important role in insulin gene transcriptional activation and islet cell formation. BETA2 is a transcription factor in the basic helix–loop–helix (bHLH) family, which activates expression of tissue-specific genes in several developmental systems. In this study, we investigated the functional and physical interactions of ISL1 and BETA2 in promoting insulin gene transcription in non-β cells. Using the luciferase assay, we demonstrated that ISL1 and BETA2 could activate insulin gene transcription synergistically. Co-immunoprecipitation also supported that ISL1 and BETA2 appear in one complex and this physical interaction mediates the synergy between these two proteins.

Keywords: ISL1; BETA2; Interaction; Insulin gene transcription

Placental cathepsin M is alternatively spliced and exclusively expressed in the spongiotrophoblast layer by Salima Bode; Christoph Peters; Jan M. Deussing (pp. 160-167).

Cathepsin M and cathepsin-3 are cysteine peptidases expressed exclusively in the murine placenta. Their expression increases continuously from 11.5 dpc until the end of gestation. The cathepsin M gene consists of 8 exons and 7 introns covering 6 kb of genomic DNA on mouse chromosome 13. Multiple variants of CTSM were identified which display alternative splicing of exon 2 or exon 7. Alternative splicing of exon 2 does not affect the translated region of CTSM whereas aberrant splicing of exon 7 will results in enzymatically inactive versions of CTSM which still might retain inhibitory activity towards cysteine peptidases. Besides two defined major transcription start sites the putative promoter region comprises of a TATA-box and a relatively low (41%) G+C content reflecting its highly specific spatial and temporal expression pattern. Similar features are found within the promoter region of CTS3 which is highly homologous to CTSM. Both cathepsin M and -3 expression are confined to the spongiotrophoblast layer of the mouse placenta an expression pattern which is unique among cysteine peptidases located within the cluster of cathepsin J-like peptidases on mouse chromosome 13.

Keywords: Cysteine peptidase; Alternative splicing; Spongiotrophoblast; Mouse placenta

C/EBP and Cdx family factors regulate liver fatty acid binding protein transgene expression in the small intestinal epithelium by Lora J. Staloch; Joyce K. Divine; Joshua T. Witten; Theodore C. Simon (pp. 168-178).

A transgene constructed from the rat liver fatty acid binding protein gene ( Fabp1) promoter is active in all murine small intestinal crypt and villus epithelial cells. Coincident Cdx and C/EBP transcription factor binding sites were identified spanning Fabp1 nucleotides −90 to −78. CDX-1, CDX-2, C/EBPα, and C/EBPβ activated the Fabp1 transgene in CaCo-2 cells, and mutagenizing the −78 site prevented activation by these factors. CDX but not C/EBP factors bound to the site in vitro, although C/EBP factors competed with CDX factors for transgene activation. The −78 site adjoins an HNF-1 site, and CDX and C/EBP family factors cooperated with HNF-1α but not HNF-1β to activate the transgene. Furthermore, CDX-1, CDX-2, C/EBPα, and C/EBPβ bound to HNF-1α and HNF-1β. The transgene with a mutagenized −78 site was silenced in vivo specifically in small intestinal crypt epithelial cells but remained active in villus cells. These results demonstrate functional interactions between HNF-1, C/EBP, and CDX family factors and suggest that these interactions may contribute to differential transcriptional regulation in the small intestinal crypt and villus compartments.

Keywords: Fabp1; CDX; C/EBP; Small intestine; Crypt; Gene expression

Identification of target genes of the transcription factor HNF1β and HNF1α in a human embryonic kidney cell line by Sabine Senkel; Bel�n Lucas; Ludger Klein-Hitpass; Gerhart U. Ryffel (pp. 179-190).

Hepatocyte nuclear factor 1beta (HNF1β, TCF2) is a tissue-specific transcription factor whose mutation in humans leads to renal cysts, genital malformations, pancreas atrophy and maturity onset diabetes of the young (MODY5). Furthermore, HNF1β overexpression has been observed in clear cell cancer of the ovary. To identify potential HNF1β target genes whose activity may be deregulated in human patients, we established a human embryonic kidney cell line (HEK293) expressing HNF1β conditionally. Using Flp recombinase, we introduced wild type or mutated HNF1β at a defined chromosomal position allowing a most reproducible induction of the HNF1β derivatives upon tetracycline addition. By oligonucleotide microarrays we identified 25 HNF1β-regulated genes. By an identical approach, we identified that the related transcription factor HNF1α (TCF1) affects only nine genes in HEK293 cells and thus is a less efficient factor in these kidney cells. The HNF1β target genes dipeptidyl peptidase 4 (DPP4), angiotensin converting enzyme 2 (ACE2) and osteopontin (SPP1) are most likely direct target genes, as they contain functional HNF1 binding sites in their promoter region. Since nine of the potential HNF1β target genes are deregulated in clear cell carcinoma of the ovary, we propose that HNF1β overexpression in the ovarian cancer participates in the altered expression pattern.

Keywords: Kidney cell; Clear cell ovarian cancer; Gene expression profile; HNF1α; HNF1β; HNF4α

Tilapia metallothionein genes: PCR-cloning and gene expression studies by Cheung; Andrew Pok Lap; Vincent Kwok Lim Lam; King Ming Chan (pp. 191-201).

Genomic PCR reactions were performed to isolate gene sequences of tilapia metallothionein (tiMT) from Oreochromis mossambicus and Oreochromis aureus. Two AP1 binding sites, four metal responsive elements, and a TATA box are the major cis-acting elements identified in the 800-bp 5′ flanking region of the tiMTs obtained in this study. The tiMT gene promoter cloned from O. aureus was characterized in vitro using PLHC-1 cell-line, a hepatocellular carcinoma of a desert topminnow ( Poecciliopsis lucida), following the administrations of Cd²⁺, Co²⁺, Cu²⁺, Ni²⁺, Pb²⁺ and Zn²⁺. Only Cd²⁺, Pb²⁺ and Zn²⁺ were able to induce the transcription of tiMT gene promoter in PLHC-1 cells in a dose-dependent manner. Zn²⁺ had the highest fold induction of tiMT gene promoter activity. Deletion mutants were tested for their abilities to drive the transcription of reporter gene following Cd²⁺ and Zn²⁺ administrations. However, Cu²⁺ and Ni²⁺ also induced the production of hepatic MT mRNA in vivo. Northern blot analysis showed that liver gave the highest fold induction of MT gene expression following the administration of heavy metal ions. These data indicated that hepatic MT mRNA level in tilapia is a potential sensitive biomarker of exposure to various metal ions including Cu²⁺, Cd²⁺, Ni²⁺, Pb²⁺, Hg²⁺ and Zn²⁺ ions.

Keywords: Abbreviations; A; absorbance (1 cm); AP1; Activator protein 1; bp; basepair(s); dd; deionized and distilled; kb; kilobase(s); GRE; glucocorticoid response element; IPTG; isopropylthio-β-; d; -galactoside; kbp; kilo base-pair; MT; metallothionein; MRE; metal regulatory element or metal responsive element; NF-IL6; nuclear factor-Interleukin 6; PCR; polymerase chain reaction; RT; reverse transcription; Sp1; Specific protein 1; TBE; tris-borate-EDTA buffer; U; unitBiomarker of exposure; Gene transcription; In vitro cell study; Heavy metals

T-DNA tagging and characterization of a cryptic root-specific promoter in Arabidopsis by C. Sivanandan; T.P. Sujatha; Anand Mohan Prasad; R. Resminath; Dhiraj R. Thakare; S.R. Bhat; Srinivasan (pp. 202-208).

From a T-DNA tagged Arabidopsis population, a line, M-57 showing GUS (β-glucuronidase) expression in the vascular regions of young roots was identified. Southern analysis revealed presence of a single T-DNA insert. Using inverse PCR, the plant sequence flanking the T-DNA insertion was cloned. The insertion was identified to be in the intergenic area between loci At4G13940 and At4G13930, coding for SAHH (S-Adenosyl-l-Homocysteine Hydrolase) and SHMT (Serine Hydroxy Methyl Transferase) genes, respectively. A 452-bp fragment immediately upstream of the T-DNA insertion when cloned and mobilized as a GUS fusion was capable of driving a similar root-specific expression of reporter gene in transgenic Arabidopsis plants and their progenies. This cryptic promoter element does not show the presence of any known root-specific promoter element.

Keywords: Arabidopsis; Root; GUS; Cryptic promoter

Involvement of upstream stimulatory factor in regulation of the mouse Prnd gene coding for Doppel protein by Janka Sepelakova; Martina Takacova; Silvia Pastorekova; Juraj Kopacek (pp. 209-214).

Promoter of the Prnd gene coding for the Prion-like protein Doppel contains two critical cis-regulatory elements, NF-Y consensus motif and canonical E-box. Here, we studied a role of the upstream stimulatory factor (USF) in the E-box-mediated activation of Prnd transcription. Co-expression of USF-1 with the luciferase reporter gene driven by the −185/+27 Prnd promoter fragment resulted in several fold increase of the luciferase activity. Conversely, mutations within the E-box led to a significantly reduced Prnd promoter activation. USF-1 binding was supported by the gel shift assay, supershift with USF-1 antibody and UV cross-linking. The activation capacity of the related USF-2, c-Myc and HIF-2α proteins was lower compared to USF-1 suggesting that USF-1 is the major E-box-binding transcription factor regulating the Prnd promoter.

Keywords: Doppel; Promoter; Gel shift; Supershift, USF; E-box

Regulatory elements of microfibril-associated glycoprotein-1 gene expression in muscle cells by Fernando Segade; Robert P. Mecham (pp. 215-224).

The Mfap2 gene encodes the microfibril-associated glycoprotein-1 (MAGP1), an extracellular matrix protein of microfibrillar structures. The gene is transcribed from a major transcription start site embedded in a CpG island. Mapping of transcriptionally active regions in the 5′ flanking sequence identified a region, located between nucleotides −339 and −109 as the Mfap2 basal promoter. Site-directed and random mutagenesis demonstrated that a KLF sequence motif at −256/−270, an E-box at −222/−229, and a GC-box at −117/−125, are critical for the promoter function. Using electrophoresis mobility shift assays, we find that the KLF motif mediates the binding of GKLF/KLF4, whereas the E-box is a target for both Upstream Stimulatory Factors 1 and 2, and the GC box at −117/−125 forms complexes with Sp1 and Sp3, but not with Sp4 or AP2α. A sequence element spanning position −150 may represent the binding motif of an uncharacterized transcription factor. The basal transcriptional regulation of Mfap2 in muscle cells is discussed.

Keywords: Abbreviations; EMSA; electrophoretic mobility shift assay; IP; immunoprecipitation; MAGP; microfibril-associated glycoprotein; TF; transcription factorMAGP1; Microfibril; Sp1; KLF4; USF; Myoblast; C2C12

Gene Structure and Expression Cumulative Contents (pp. 225-226).

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BBA - Gene Structure and Expression (v.1731, #3)