Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression最新文献

A novel zinc transporter has been purified and cloned from rat renal brush border membrane. This transporter was designated as Zip10 encoded by Slc39a10 gene and characterized as zinc importer. Present study documents the impact of thyroid hormones on the expression of Zip10 encoded by Slc39a10 gene in rat model of hypo and hyperthyroidism. Serum T₃ and T₄ levels were reduced significantly in hypothyroid rats whereas these levels were significantly elevated in hyperthyroid rats as compared to euthyroid rats thereby confirming the validity of the model. Kinetic studies revealed a significant increase in the initial and equilibrium uptake of Zn⁺⁺ in both intestinal and renal BBMV of hyperthyroid rats in comparison to hypothyroid and euthyroid rats. By RT-PCR, Slc39a10 mRNA expression was found to be significantly decreased in hypothyroid and increased in hyperthyroid as compared to euthyroid rats. These findings are in conformity with the immunofluorescence studies that revealed markedly higher fluorescence intensity at periphery of both intestinal and renal cells isolated from hyperthyroid rats as compared to hypothyroid and euthyroid rats. Higher expression of Zip10 protein in hyperthroid group was also confirmed by western blot. These findings suggest that expression of zinc transporter protein Zip10 (Slc39a10) in intestine and kidney is positively regulated by thyroid hormones.

Geraniol 10-hydroxylase (G10H) is an important enzyme in the biosynthetic pathway of monoterpenoid alkaloids found in diverse plant species. The Catharanthus roseus G10H controls the first committed step in biosynthesis of terpenoid indole alkaloids (TIA). The C. roseus G10H promoter sequence was isolated by a PCR-based genome walking method. Sequence analysis revealed that the G10H promoter contains several potential eukaryotic regulatory elements involved in regulation of gene expression. The major transcription start site of the promoter was mapped to an adenine 31 bp downstream of the TATA-box. For functional characterization, transcriptional fusions between the G10H promoter fragments with 5′ or 3′ deletions and the GUS reporter gene were generated and their expressions were analyzed in a tobacco protoplast transient expression assay. Deletion of the promoter down to − 318 bp had little effect on GUS activity. However, further deletion of the promoter to position − 103 resulted in approximately 5-fold reduction of GUS activity. Gain-of-function experiments revealed the presence of three potential transcriptional enhancers located in regions between − 191 and − 147, − 266 and − 188, and − 318 and − 266, respectively. The G10H promoter was capable of conferring stable GUS expression in transgenic tobacco plants and C. roseus hairy roots. In transgenic tobacco seedlings GUS expression was tissue-specific, restricted to leaf and actively growing cells around the root tip, and not detected in the hypocotyls, root cap and older developing areas of the root. The GUS expression in both transgenic C. roseus hairy roots and tobacco seedlings were responsive to fungal elicitor and methyljasmonate. Compared to other known promoters of TIA pathway genes, the G10H promoter contains unique binding sites for several transcription factors, suggesting that the G10H promoter may be regulated by a different transcriptional cascade.

Intestinal epithelial cells respond to inflammatory extracellular stimuli by activating mitogen activated protein kinase (MAPK) signaling, which mediates numerous pathophysiological effects, including intestinal inflammation. Here, we show that a novel isoform of SPS1-related proline alanine-rich kinase (SPAK/STE20) is involved in this inflammatory signaling cascade. We cloned and characterized a SPAK isoform from inflamed colon tissue, and found that this SPAK isoform lacked the characteristic PAPA box and alphaF loop found in SPAK. Based on genomic sequence analysis the lack of PAPA box and alphaF loop in colonic SPAK isoform was the result of specific splicing that affect exon 1 and exon 7 of the SPAK gene. The SPAK isoform was found in inflamed and non-inflamed colon tissues as well as Caco2-BBE cells, but not in other tissues, such as liver, spleen, brain, prostate and kidney. In vitro analyses demonstrated that the SPAK isoform possessed serine/threonine kinase activity, which could be abolished by a substitution of isoleucine for the lysine at position 34 in the ATP-binding site of the catalytic domain. Treatment of Caco2-BBE cells with the pro-inflammatory cytokine, interferon γ, induced expression of the SPAK isoform. Over-expression of the SPAK isoform in Caco2-BBE cells led to nuclear translocation of an N-terminal fragment of the SPAK isoform, as well as activation of p38 MAP kinase signaling cascades and increased intestinal barrier permeability. These findings collectively suggest that pro-inflammatory cytokine signaling may induce expression of this novel SPAK isoform in intestinal epithelia, triggering the signaling cascades that govern intestinal inflammation.

The insulin promoter contains a number of dissimilar cis-acting regulatory elements that bind a range of tissue specific and ubiquitous transcription factors. Of the regulatory elements within the insulin promoter, the cyclic AMP responsive element (CRE) binds by far the most diverse array of transcription factors. Rodent insulin promoters have a single CRE site, whereas there are four CREs within the human insulin gene, of which CRE2 is the only one conserved between species. The aim of this study was to characterise the human CRE2 site and to investigate the effects of the two principal CRE-associated transcription factors; CREB-1 and ATF-2. Co-transfection of INS-1 pancreatic β-cells with promoter constructs containing the human insulin gene promoter placed upstream of the firefly luciferase reporter gene and expression plasmids for ATF-2 or CREB-1 showed that ATF-2 stimulated transcriptional activity while CREB-1 elicited an inhibitory effect. Mutagenesis of CRE2 diminished the effect of ATF-2 but not that of CREB-1. ATF-2 was shown to bind to the CRE2 site by electrophoretic mobility shift assay and by chromatin immunoprecipitation, while siRNA mediated knockdown of ATF-2 diminished the stimulatory effects of cAMP related signalling on promoter activity. These results suggest that ATF-2 may be a key regulator of the human insulin promoter possibly stimulating activity in response to extracellular signals.

The coding region of c-myc mRNA encompassing the coding region determinant (CRD) nucleotides (nts) 1705–1792 is critical in regulating c-myc mRNA stability. This is in part due to the susceptibility of c-myc CRD RNA to attack by an endoribonuclease. We have previously purified and characterized a mammalian endoribonuclease that cleaves c-myc CRD RNA in vitro. This enzyme is tentatively identified as a 35 kDa RNase1-like endonuclease. In an effort to understand the sequence and secondary structure requirements for RNA cleavage by this enzyme, we have determined the secondary structure of the c-myc CRD RNA nts 1705–1792 using RNase probing technique. The secondary structure of c-myc CRD RNA possesses five stems; two of which contain 4 base pairs (stems I and V) and three consisting of 3 base pairs (stems II, III, and IV). Endonucleolytic assays using the c-myc CRD and several c-myc CRD mutants as substrates led to the following conclusions: (i) the enzyme prefers to cleave in between the dinucleotides UA, CA, and UG in single-stranded regions; (ii) the enzyme is more specific towards UA dinucleotides. These properties further distinguish the enzyme from previously described mammalian endonuclease that cleaves c-myc mRNA in vitro.

The P-type Mg²⁺-ATPase, termed ATPase II (Atp8a1), is a putative aminophospholipid transporting enzyme, which helps to maintain phospholipid asymmetry in cell membranes. In this project we have elucidated the organization of the mouse ATPase II gene and identified its promoter. Located within chromosome 5, this gene spans about 224 kb and consists of 38 exons, three of which are alternatively spliced (exons 7, 8 and 16), giving rise to two transcript variants. Translation of these transcripts results in two ATPase II isoforms (1 and 2) composed of 1164 and 1149 amino acids, respectively. Using RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) we identified multiple transcription start sites (TSS) in messages obtained from heart, lung, liver, and spleen. The mouse ATPase II promoter is TATA-less and lacks a consensus initiator sequence. Luciferase reporter analysis of full and core promoters revealed strong activity and little cell type specificity, possibly because more flanking, regulatory sequences are required to cause such tissue specificity. In the neuronal HN2, N18, SN48 cells and the NIH3T3 fibroblast cells, but not in the B16F10 melanoma cells, the core promoter (− 318/+ 193 with respect to the most common TSS) displayed significantly higher activity than the full promoter (− 1026/+ 193). Serial 5′ deletion of the core promoter revealed significant cell type-specific activity of the fragments, suggesting differential expression and use of transcription factors in the five cell lines tested. Additionally distribution of the TSS was organ specific. Such observations suggest tissue-specific differences in transcription initiation complex assembly and regulation of ATPase II gene expression. Information presented here form the groundwork for further studies on the expression of this gene in apoptotic cells.

RNA interference (RNAi) is implicated in maintaining tandem DNA arrays as constitutive heterochromatin. We used chromatin immunoprecipitation with antibodies to RNA polymerase II (RNAPol-ChIP) to test for transcription of the following repeat arrays in human cells: subtelomeric D4Z4, pericentromeric satellite 2, and centromeric satellite α. D4Z4 has a promoter-like sequence upstream of an ORF in its 3.3-kb repeat unit. A short D4Z4 array at 4q35 is linked to facioscapulohumeral muscular dystrophy (FSHD). By RNAPol-ChIP and RT-PCR, little or no transcription of D4Z4 was detected in FSHD and normal myoblasts; lymphoblasts from an FSHD patient, a control, and a patient with D4Z4 hypomethylation due to mutation of DNMT3B (ICF syndrome); and normal or cancer tissues. However, RNAPol-ChIP assays indicated transcription of D4Z4 in a chromosome 4-containing human–mouse somatic cell hybrid. ChIP and RT-PCR showed satellite DNA transcription in some cancers and lymphoblastoid cell lines, although only at a low level. Given the evidence for the involvement of RNAi in satellite DNA heterochromatinization, it is surprising that, at most, a very small fraction of satellite DNA was associated with RNA Pol II. In addition, our results do not support the previously hypothesized disease-linked differential transcription of D4Z4 sequences in short, FSHD-linked arrays.

Members of the Caenorhabditis elegans REF-1 family of bHLH proteins are atypical in that each protein contains two bHLH domains. In this study we describe a functional and molecular characterization of the REF-1 family members, hlh-29/hlh-28. 5′-RACE results confirm the presence of two bHLH domain coding regions in a single transcript and quantitative PCR (qPCR) shows that hlh-29/hlh-28 mRNA is detected in wild-type animals throughout development. A promoter fusion of hlh-29 to the green fluorescent protein shows post-embryonic reporter activity in cells of the vulva, the somatic gonad, the intestine and in neuronal cells of the head and tail. Loss of hlh-29/hlh-28 function via RNA interference (RNAi) results in multiple phenotypes including late embryonic lethality, yolk protein accumulation, everted vulva, bordering behavior, and alter chemosensory responses.

Solution structures of DNA/RNA hybrid duplexes, d(GCGCA*AA*ACGCG): r(cgcguuuugcg)d(C) (designated PP57), containing two C8-propynyl 2′-deoxyadenosines (A*) and unmodified hybrid (designated U4A4) are solved. The C8-propynyl groups on 2′-deoxyadenosine perturb the local structure of the hybrid duplex, but overall the structure is similar to that of canonical DNA/RNA hybrid duplex except that Hoogsteen hydrogen bondings between A* and U result in lower thermal stability. RNase H is known to cleave RNA only in DNA/RNA hybrid duplexes. Minor groove widths of hybrid duplexes, sugar puckerings of DNA are reported to be responsible for RNase H mediated cleavage, but structural requirements for RNase H mediated cleavage still remain elusive. Despite the presence of bulky propynyl groups of PP57 in the minor groove and greater flexibility, the PP57 is an RNase H substrate. To provide an insight on the interactions between RNase H and substrates we have modeled Bacillus halodurans RNase H-PP57 complex, our NMR structure and modeling study suggest that the residue Gly(15) and Asn(16) of the loop residues between first β sheet and second β sheet of RNase HI of Escherichia coli might participate in substrate binding.