ATTTA Motifs Research Articles

Genomic characterization and transcription analysis of European sea bass (Dicentrarchus labrax) rtp3 genes

Fish RTP3, belonging to the receptor-transporting protein family, display several functions, including a putative antiviral role as virus-responsive gene. In this work, we have identified and characterized two different European sea bass rtp3 genes. In addition, an in vivo transcription analysis in response to LPS, poly I:C and betanodavirus infection (RGNNV genotype) has been performed. The sequence analysis showed that European sea bass displays two rtp3 genes, X1 and X2, composed of two exons and a single intron (1007-bp and 888-bp long, respectively), located within the ORF sequence. The full-length cDNA is 1969 bp for rtp3 X1, and 1491 bp for rtp3 X2. Several ATTTA motifs have been found in the intron sequence of both genes, whereas rtp3 X1 also contains this motif in both untranslated regions. The transcription analyses revealed significant level of rtp3 X2 mRNA in brain and head kidney after LPS and poly I:C inoculation; however, the induction elicited by RGNNV infection was much higher, suggesting an essential role for this protein in controlling NNV infections.

Expression and functional analysis of CsA-IPT5 splice variants during shoot branching in Camellia sinensis.

Alternative splicing (AS) is a process by which several functional splice variants are generated from the same precursor mRNA. In our recent study, five CsA-IPT5 splice variants with various numbers of ATTTA motifs in the untranslated regions (UTRs) were cloned. Meanwhile, their transient expression, as well as the expression and functional analysis in the two shoot branching processes were studied. Here, we examined how these splice variants regulate the other three important shoot branching processes, including the spring tea development, the distal branching of new shoots, and the shoot branching induced by 2,3,5-triiodobenzoic acid (TIBA) spraying, and thus unraveling the key CsA-IPT5 transcripts which play the most important roles in the shoot branching of tea plants. The results showed that the increased expression of 5′ UTR AS3, 3′ UTR AS1 and 3′ UTR AS2 could contribute to the increased synthesis of tZ/iP-type cytokinins (CKs), thus promoting the spring tea development. Meanwhile, in the TIBA-induced shoot branching or in the distal branching of the new shoots, CsA-IPT5 transcripts regulated the synthesis of CsA-IPT5 protein and CKs through transcriptional regulation of the ratios of its splice variants. Moreover, 3′ UTR AS1 and 3′ UTR AS2 both play key roles in these two processes. In summary, it is revealed that 3′ UTR AS1 and 3′ UTR AS2 of CsA-IPT5 might act as the predominant splice variants in shoot branching of the tea plant, and they both can serve as gene resources for tea plant breeding.

A novel adenylate isopentenyltransferase 5 regulates shoot branching via the ATTTA motif in Camellia sinensis

BackgroundShoot branching is one of the important agronomic traits affecting yields and quality of tea plant (Camellia sinensis). Cytokinins (CTKs) play critical roles in regulating shoot branching. However, whether and how differently alternative splicing (AS) variant of CTKs-related genes can influence shoot branching of tea plant is still not fully elucidated.ResultsIn this study, five AS variants of CTK biosynthetic gene adenylate isopentenyltransferase (CsA-IPT5) with different 3′ untranslated region (3ˊ UTR) and 5ˊ UTR from tea plant were cloned and investigated for their regulatory effects. Transient expression assays showed that there were significant negative correlations between CsA-IPT5 protein expression, mRNA expression of CsA-IPT5 AS variants and the number of ATTTA motifs, respectively. Shoot branching processes induced by exogenous 6-BA or pruning were studied, where CsA-IPT5 was demonstrated to regulate protein synthesis of CsA-IPT5, as well as the biosynthesis of trans-zeatin (tZ)- and isopentenyladenine (iP)-CTKs, through transcriptionally changing ratios of its five AS variants in these processes. Furthermore, the 3′ UTR AS variant 2 (3AS2) might act as the predominant AS transcript.ConclusionsTogether, our results indicate that 3AS2 of the CsA-IPT5 gene is potential in regulating shoot branching of tea plant and provides a gene resource for improving the plant-type of woody plants.

Different origins of paralogues of salmonid TNR1 and TNFR2: Characterisation and expression analysis of four TNF receptor genes in rainbow trout Oncorhynchus mykiss

Mammalian TNFR1 and TNFR2 bind TNFα and TNFβ, and provide key communication signals to a variety of cell types during development and immune responses that are crucial for cell survival, proliferation and apoptosis. In teleost fish TNFβ is absent but TNFα has been expanded by the third whole genome duplication (3R WGD) and again by a 4R WGD in some lineages, leading to the four TNFα paralogues known in salmonids. Two paralogues for each of TNFR1 and TNFR2 have been cloned in rainbow trout in this study and are present in other salmonid genomes. Whilst the TNFR2 paralogues were generated via the 4R salmonid WGD, the TNFR1 paralogues arose from a local en bloc duplication. Functional diversification of TNFR paralogues was evidenced by differential gene expression and modulation, upstream ATGs affecting translation, ATTTA motifs in the 3′-UTR regulating mRNA stability, and post-translational modification by N-glycosylation. Trout TNFR are highly expressed in immune tissues/organs, and other tissues, in a gene- and tissue-specific manner. Furthermore, their expression is differentially modulated by PAMPs and cytokines in a cell type- and stimulant-specific manner. Such findings suggest an important role of the TNF/TNFR axis in the immune response and other physiological processes in fish.

Tandem duplication of two tRNA genes in the mitochondrial genome of Tagiades vajuna (Lepidoptera: Hesperiidae)

To explore the debated phylogenetic relationship of two Hesperiidae subfamilies, Pyrginae and Eudaminae, and contribute to the understanding of the evolution of mitogenomic architecture in butterflies, we sequenced the complete mitogenome of Tagiades vajuna. The mitogenome is a typical circular duplex molecule of 15,359 bp. Apart from the standard 22 tRNAs, it has a tandem duplication of trnS(AGN) and trnE, which is unique in lepidopteran insects. Comparison with Ctenoptilum vasava indicates that the trnS1 duplication is not an ancestral state shared with other species of Tagiadini. Independent origin of the trnS1 duplications was further confirmed by the reconstruction of the ancestral character state based on the topology of the phylogram. Furthermore, comparative analysis of mitogenomes with and without tRNA duplications indicates that tRNA duplication does not alter the codon usage pattern. The mitogenome has negative AT- and GC-skews, and it is highly A+T-biased (79.7%). The AT-rich (or control) region (283 bp) contains ATAGA and ATTTA motifs. Regarding the phylogenetic analysis, we found that removal of the third codon position (3CP) from datasets used for the mitochondrial phylogenomics of Hesperiidae is likely to produce results that are more consistent: Pyrginae were rendered paraphyletic by Eudaminae in both analyses of the dataset from which the 3CP was removed (13 PCGs + all RNAs), but inclusion of the 3CP resulted in a destabilized topology, resulting in both monophyly and polyphyly. We conclude that even shallow-phylogenies of insects should pay close attention to compositional and mutational biases in mitogenomes.

Genetic characterization and transcription analyses of the European sea bass (Dicentrarchus labrax) isg15 gene.

Fish interferons are cytokines involved in its resistance to viral infections by inducing the transcription of several interferon-induced genes, such as isg15. The aim of the present study was the genetic characterization of the European sea bass isg15 gene, describing the regulatory motifs found in its sequence. In addition, an invivo analysis of transcription in response to betanodavirus (RGNNV genotype) and poly I:C has been performed. The analysis of the resulting sequences showed that sea bass isg15 gene is composed of two exons and a single 276-bp intron located at the 5'-UTR region. The full length cDNA is 1143-bp, including a 102-bp 5'-UTR region, a 474-bp ORF, and a 291-bp 3'-UTR region. Several mRNA-regulatory elements, including three unusual ATTTA instability motifs in the intron, and four ATTTA motifs along with a cytoplasmic polyadenylation element in the 3'-UTR region, have been found in this sequence. The invivo analyses revealed a similar kinetics and level of transcription in fish brain and head kidney after poly I:C inoculation; however, the induction caused by RGNNV started earlier in brain, where the upregulation of isg15 gene transcription was high. The present study contributes to further characterize the European sea bass IFN I response against RGNNV infections.

Identification and characterisation of the IL-27 p28 subunits in fish: Cloning and comparative expression analysis of two p28 paralogues in Atlantic salmon Salmo salar

Interleukin (IL)-27 is an IL-6/IL-12 family member with pro-inflammatory and anti-inflammatory properties. It is a heterodimeric cytokine composed of an α-chain p28 and a β-chain Ebi3 (Epstein–Barr virus induce gene 3). The p28 subunit can also be secreted as a monomer and function as IL-30 that acts as an inhibitor of IL-27 signalling. At present, the p28 gene has only been described in mammals. Thus, for the first time outwith mammals, we have identified seven p28 molecules in six divergent teleost fish species, Atlantic salmon, two cichlids, two cyprinids and a yellowtail. The fish p28 molecules have higher similarities to mammalian p28 than other IL-6/12 family members. Critical residues involved in the interaction with Ebi3 and the receptor gp130 are highly conserved. The prediction that these are true orthologues is supported by phylogenetic and synteny analysis.Two p28 paralogues (p28a and p28b) sharing 72% aa identity have been identified and characterised in Atlantic salmon. There are multiple upstream ATGs in the 5′-UTR and ATTTA motifs in the 3′-UTR of both cDNA sequences, suggesting regulation at the post-transcriptional and translational level. Both salmon p28 genes are highly expressed in immune relevant tissues, such as thymus, gills, spleen and head kidney. Conversely salmon Ebi3 is highly expressed in other organs, such as liver and caudal kidney. The expression of p28 but not Ebi3 was induced by PAMPs and recombinant cytokines in head kidney cells, and in spleen by Poly I:C challenge in vivo. The dissociation of the expression and modulation of p28 and Ebi3 suggest that both p28 and Ebi3 may be secreted alone or with other partners.

Molecular Cloning and Functional Characterization of the Dual Oxidase (BmDuox) Gene from the Silkworm Bombyx mori

Reactive oxygen species (ROS) from nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and their related dual oxidases are known to have significant roles in innate immunity and cell proliferation. In this study, the 5,545 bp cDNA of the silkworm Bombyx mori dual oxidase (BmDuox) gene containing a full-length open reading frame was cloned. It was shown to include an N-terminal signal peptide consisting of 28 amino acid residues, a 240 bp 5′-terminal untranslated region (5′-UTR), an 802 bp 3′-terminal region (3′-UTR), which contains nine ATTTA motifs, and a 4,503 bp open reading frame encoding a polypeptide of 1,500 amino acid residues. Structural analysis indicated that BmDuox contains a typical peroxidase domain at the N-terminus followed by a calcium-binding domain, a ferric-reducing domain, six transmembrane regions and binding domains for flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD). Transcriptional analysis revealed that BmDuox mRNA was expressed more highly in the head, testis and trachea compared to the midgut, hemocyte, Malpighian tube, ovary, fat bodies and silk glands. BmDuox mRNA was expressed during all the developmental stages of the silkworm. Subcellular localization revealed that BmDoux was present mainly in the periphery of the cells. Some cytoplasmic staining was detected, with rare signals in the nucleus. Expression of BmDuox was induced significantly in the larval midgut upon challenge by Escherichia coli and Bombyx mori nucleopolyhedrovirus (BmNPV). BmDuox-deleted larvae showed a marked increase in microbial proliferation in the midgut after ingestion of fluorescence-labeled bacteria compared to the control. We conclude that reducing BmDuox expression greatly increased the bacterial load, suggesting BmDuox has an important role in inhibiting microbial proliferation and the maintenance of homeostasis in the silkworm midgut.

Molecular cloning and expression profiles of nitric oxide synthase (NOS) in mud crab Scylla paramamosain

The importance of the nitric oxide synthase (NOS) gene family is demonstrated by many studies in vertebrates and invertebrates in recent years. However, it keeps unknown of nitric oxide (NO) system and NOS gene family in mud crab Scylla paramamosain, an important cultured commercial crustacean in China and Pacific area. In this report, the cDNA of NOS containing full-length ORF was cloned from mud crab, S. paramamosain. It was of 4424 bp, including a 5′-terminal untranslated region (UTR) of 239 bp, a 3′-terminal UTR of 540 bp, which contained two ATTTA motifs, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids. Structural analysis indicated that NOS contained a typical NO synthase domain at the N-terminal, next to a flavodoxin 1 domain, a flavin adenine dinucleotide (FAD) binding domain, respectively, and a conservative nicotinamide adenine dinucleotide (NAD) binding domain structure at the C-terminal. Quantitative real-time PCR analysis revealed S. paramamosain NOS (SpNOS) to be expressed in all tissues examined, with the highest expression in midintestine and the weakest level in heart and eyestalk. The expression profiles of SpNOS indicated that the NOS expression levels were significantly induced in midintestine, hepatopancrease and hemocytes after challenged with Vibrio Parahaemolyticus, the synthetic double-stranded RNA polyinosinic polycytidylic acid (poly I:C) and lipopolysaccharides (LPS). The NOS activity in hemocytes showed significant increase during at 24 h–48 h time period after immune challenges with V. Parahaemolyticus, poly I:C and LPS. Results here may suggest that the inducible NOS play an important role in mud crab’s defense against pathogenic infection.

Extremely high conservation in the untranslated region as well as the coding region of CNP mRNAs throughout elasmobranch species

C-type natriuretic peptide (CNP) is a crucial osmoregulatory hormone in elasmobranchs, participating in salt secretion and drinking. In contrast to teleosts and tetrapods in which the NP family is composed of a group of structurally related peptides, we have shown that CNP is the sole NP in sharks. In the present study, CNP cDNAs were cloned from four species of batoids, another group of elasmobranchs. The cloned batoid CNP precursors contained a plausible mature peptide of 22 amino acid residues that is identical to most shark CNP-22s, but five successive amino acids were consistently deleted in the prosegment compared with shark precursors, supporting the diphyletic classification of sharks and rays. In addition, molecular phylogenetic trees of CNP precursors were consistent with a diphyletic interpretation. Except for the deletion, the nucleotide and deduced amino acid sequences of the CNP cDNAs are extremely well-conserved among all elasmobranch species, even between sharks and rays. Surprisingly, high conservation is evident not only for the coding region, but also for the untranslated regions. It is most likely that the high conservation is due to the low nucleotide substitution rate in the elasmobranch genome, and high selection pressure. The 3′-untranslated region of the elasmobranch CNP cDNAs contained three to six repeats of the ATTTA motif that is associated with the regulation of mRNA stability and translation efficiency. Alternative polyadenylation sites were also found; the long 3′-untranslated region contains a core of ATTTA motifs while the short form has only one or no ATTTA motif, indicating that the post-transcriptional modification of mRNA is important for regulation of CNP synthesis. These characteristics in the 3′-untranslated region were conserved among all elasmobranch CNP cDNAs. Since CNP has been implicated as a fast-acting hormone to facilitate salt secretion from the rectal gland, the conserved 3′-untranslated region most likely contributes to rapid regulation of CNP synthesis in elasmobranchs in response to acute changes in internal and external environments.

Characterization and functional significance of myotrophin: A gene with multiple transcripts

The underlying mechanism for the development of cardiac hypertrophy that advances to heart failure is not known. Many factors have been implied to play a role in this process. Among others, we have isolated and identified myotrophin, a factor that stimulates myocytes growth, from spontaneously hypertensive rat (SHR) heart and patients with dilated cardiomyopathy. The gene encoding myotrophin has been cloned and expressed in E. coli. Recently, myotrophin gene has been mapped and shown to be a novel gene localized in human chromosome 7q-33. To define the characteristics of each transcript and its pathophysiological significance, we examined transcripts of myotrophin in SHR heart during progression of hypertrophy. Northern blot analysis of myotrophin mRNA showed multiple transcripts. We isolated and characterized various myotrophin cDNA clones corresponding to the multiple transcripts by 5′ “stretch plus” rat heart cDNA library screening. Sequence analysis of these cDNA clones indicates that each clone has a unique 5′ UTR and multiple 3′ UTR with varying lengths, repeated ATTTA motifs and many polyadenylation signals. In vitro transcripts generated from all these myotrophin-specific cDNA clones translate in vitro to a 12-kD protein. Among pathophysiological significance, we determined mRNA expression in 9 days old, 3 weeks old and 31 weeks old and observed a linear increased during the progression of hypertrophy. In WKY, this mRNA level remained the same throughout the growth and development of hypertrophy. Our data strongly suggest that myotrophin appears to be a candidate gene for cardiac hypertrophy and heart failure.

Cloning of the genes for the pituitary glycoprotein hormone alpha and follicle-stimulating hormone beta subunits in the Japanese crested ibis, Nipponia nippon.

We have isolated a part of the gene for the pituitary glycoprotein hormone common alpha subunit (PGHalpha) and the whole gene for the follicle-stimulating hormone beta subunit (FSHbeta) in the Japanese crested ibis (Nipponia nippon), a critically endangered bird species in East Asia. The nucleotide sequence of a part of the PGHalpha gene (5026 bp) contained three exons holding the whole coding and 3' untranslated regions, but lacked a 5' untranslated region. Its exon-intron structure was similar to that in mammals, but different from that in teleosts in the location of the second intron. For the FSHbeta gene, the nucleotide sequence of 7633 bp was assembled from two phage clones. The exon-intron structure of three exons and two introns was similar to that observed in mammals and teleosts. In the putative promoter region of the ibis FSHbeta gene, a progesterone responsive element (PRE)-like sequence and two AP-1 responsive element-like sequences reported in the ovine FSHbeta gene were not conserved in complete form. The increased number of ATTTA motifs in the putative 3' untranslated region in comparison with those in Japanese quail and chicken FSHbeta cDNA suggested that more rapid degradation of FSHbeta mRNA occurs in this species. Deduced amino acid sequences of the ibis PGHalpha and FSHbeta showed high similarities with those of the corresponding subunits of other avian species. This is the first report on the genomic sequences of the PGHalpha and FSHbeta in an avian species.

A Type 2 Diabetes-Associated Polymorphic ARE Motif Affecting Expression of PPP1R3 Is Involved in RNA–Protein Interactions

We have previously described a polymorphism in the 3′ untranslated region (UTR) of the PPP1R3 gene that encodes the muscle-specific glycogen-targeting regulatory PP1 subunit. This polymorphism alters the distance between two putative mRNA-destabilizing ATTTA (AUUUA) motifs and is distinguished by a 10-nucleotide (allele ARE1) vs a 2-nucleotide interval (allele ARE2). ARE2 is associated with insulin resistance as well as increased prevalence of type 2 diabetes in the Pima Indians, and correlates with reduced expression of this subunit in vivo, causing a 10-fold half-life reduction of reporter mRNA in NIH3T3 cells. Gel shift assays, Northwestern blotting, and RNA–protein UV crosslinking revealed three proteins (43, 80, and 139 kDa) binding to the polymorphic ARE region in these cells. The interactions are sequence specific, and can be suppressed by an unlabeled competitor in a dose-dependent manner. The less stable ARE2 allele shows at least 2-fold higher relative protein binding, indicating that the polymorphic ARE region has a mRNA-destabilizing role. We suggest that the increased protein binding to ARE2 contributes to a faster degradation of PPP1R3 mRNA carrying this allele, and the resulting lower concentration of the protein contributes to insulin resistance, thus increasing the risk for development of type 2 diabetes.

MOLECULAR CLONING OF INTERLEUKIN 1β FROM RAINBOW TROUT ONCORHYNCHUS MYKISS REVEALS NO EVIDENCE OF AN ICE CUT SITE

The complete coding sequence of rainbow trout IL-1β has been obtained. The gene contains a short 5′ UTR (97bp), a 780bp open reading frame and a 466bp 3′ UTR, which includes a polyadenylation signal, 7 ATTTA motifs and an 18bp poly A tail. The predicted amino acid sequence (260 amino acids) contains 3 potential glycosylation sites, with a predicted molecular weight of 29kDa, and shows between 49 and 56% amino acid similarity to mammalian IL-1βs and 57% similarity to carp IL-1β. Greatest homology was apparent within the secondary structure of the gene, with few of the amino acids known to bind to the IL-1 receptor being conserved. No ICE cut site was apparent but multiple alignment with mammalian sequences allowed a putative mature peptide of 166 amino acids to be identified, in which Ala95would be the amino terminus. Northern blot analysis showed that whilst no IL-1β expression was detectable in head kidney leukocytes immediately after isolation, expression could be induced by stimulation with LPS for 4h in culture. Similarly, with isolated head kidney macrophages expression was significantly increased following stimulation with LPS.

Bacterial Expression, Purification, and Characterization of a Novel Mouse Sulfotransferase That Catalyzes the Sulfation of Eicosanoids

Analysis of the nucleotide sequence of a recently cloned mouse sulfotransferase cDNA (clone 679153) revealed the presence in its 3′-untranslated sequence of an AT-rich region which contains four ATTTA motifs and an TTATTTAT-like sequence, commonly found among those encoding inflammation-related proteins. The recombinant enzyme expressed inEscherichia coliand purified to near electrophoretic homogeneity displayed strong sulfotransferase activities toward various prostaglandins, thromboxane B2, and leukotriene E4. These results mark the first discovery of the sulfation of eicosanoids catalyzed by a distinct sulfotransferase.

SYMPOSIUM: Experimental Biology 1995 Role of Mesangial Cell Ion Transport in Glomerular Physiology and Disease: REGULATION OF RAT KIDNEY MESANGIAL CELL PHOSPHOLIPASE A2

1. The precursor of eicosanoids is arachidonic acid, which emanates from the cleavage of the sn-2 position of phospholipids by phospholipase A2 (PLA2). Eicosanoids have diverse physiological and pathophysiological effects in the kidney. The regulation of phospholipase A2 has important implications for kidney function. 2. In the current communication we focus our attention on mesangial cell cytosolic PLA2 (cPLA2) and its regulation at the post-translational and post-transcriptional level. 3. At the post-translational level, using site directed mutagenesis of cPLA2 and a dominant negative ras, we have demonstrated that cPLA2 can be phosphorylated by mitogen activated protein (MAP-2) kinase leading to increased cPLA2 enzymatic activity. 4. At the post-transcriptional level we show that the half-life of cPLA2 mRNA in mesangial cells is significantly increased when mesangial cells are stimulated by mitogens. We further demonstrate the presence of three ATTTA motifs in the 3' untranslated region (3' UTR) of the cPLA2 cDNA. 5. Using chimeric constructs bearing the 3' UTR from rat cPLA2 fused downstream of the luciferase reporter, we demonstrate that this region exerts a destabilizing effect on cPLA2. 6. We have isolated and mapped genomic DNA and polymorphic markers for cPLA2 in the human and rat.

Cytosolic phospholipase A2 gene expression in rat mesangial cells is regulated post-transcriptionally

Cytosolic phospholipase A2 (cPLA2) is thought to be the rate-limiting enzyme in the arachidonic acid/eicosanoid cascade. The ability of various agonists to increase steady-state cPLA2 mRNA levels has previously been reported. The current study delineates the contributions of transcriptional and post-transcriptional processes to the regulation of cPLA2 gene expression in response to a variety of agonists in cultured rat glomerular mesangial cells. Epidermal growth factor, platelet-derived growth factor, serum and phorbol myristate acetate all increase the half-life of cPLA2 mRNA transcripts, indicating a role for post-transcriptional modulation of gene expression. The presence of three ATTTA motifs in the 3' untranslated region (3'UTR) of the rat cPLA2 cDNA is ascertained. Heterologous expression of chimeric constructs with different 3'UTRs ligated into the 3' end of the luciferase coding region reveals that the presence of the cPLA2 3'UTR results in reduced luciferase activity compared with constructs without the cPLA2 3'UTR. Furthermore, the luciferase activity in the constructs with the cPLA2 3'UTR is increased in response to the same agonists which stabilize endogenous cPLA2 mRNA. A negligible effect of these agonists on transcriptional control of cPLA2 is evident using promoter-reporter constructs expressed in transient and stable transfectants. Taken together, these results indicate predominant post-transcriptional regulation of cPLA2 mRNA levels.

Genomic organization, nucleotide sequence, biophysical properties, and localization of the voltage-gated K+ channel gene KCNA4/Kv1.4 to mouse chromosome 2/human 11p14 and mapping of KCNC1/Kv3.1 to mouse 7/human 11p14.3-p15.2 and KCNA1/Kv1.1 to human 12p13.

A genomic clone encoding the Shaker-related potassium channel gene, Kcna4/mKv1.4, was isolated from mice. Its coding region is contained in a single exon, encodes a protein of 654 amino acids, and shares approximately 91% nucleotide sequence identity with human KCNA4/hKv1.4. We show that 0.8 kb of the 5' noncoding region (NCR), the entire protein coding region (approximately 2.0 kb), and all of the known 3' NCR (approximately 1.1 kb) are contained within a single exon; the remaining 0.5 kb of the 5' NCR is separated from this exon by a 3.4-kb intron. The sequenced genomic region thus accounts for essentially all of the longest known transcript (4.5 kb), although the precise ends of this transcript have not been defined. The 3' NCR contains several ATTTA and ATTTG motifs that are thought to destabilize mRNAs, and these are also present in rat, bovine, and human Kcna4/Kv1.4 cDNAs. It also contains three conserved polyadenylation signals, alternate utilization of which could generate mRNAs of differing stabilities. The 5' NCR of Kcna4/mKv1.4 may also serve to regulate channel expression. This region is approximately 85% identical to KCNA4/hKv1.4 and contains eight consensus translation start sites [(G, A)NNATG] that, based on the 5'-3' scanning model, would lead to a lowering of translational efficiency. The shortest Kcna4/Kv1.4 transcript (2.4 kb) can contain at most 400 bp of NCR and should lack the 3' ATTTAs and most of the 5' ATGs; this transcript might therefore exhibit increased stability and translational efficiency. The Kcna4/mKv1.4 channel exhibited biophysical and pharmacological properties indistinguishable from its rat and human homologues. Kcna4/mKv1.4 lies on mouse chromosome 2, near the Fshb locus, and in humans on the proximal half of chromosome 11p14 near human FSHB. Another K+ channel gene, Kcnc1/mKv3.1, lies approximately 1.8 cM from the Myod-1 gene on mouse chromosome 7, and in situ hybridization localizes KCNC1/hKv3.1 to the homologous region on human chromosome 11p14.3-p15.2. A third gene, KCNA1/hKv1.1, was mapped to human 12p13.

Regulation of collagenase gene expression by IL-1 beta requires transcriptional and post-transcriptional mechanisms.

Interleukin-1 beta is believed to contribute to the pathophysiology of rheumatoid arthritis by activating collagenase gene expression. We have used a cell culture model of rabbit synovial fibroblasts to examine the molecular mechanisms of IL-1 beta-mediated collagenase gene expression. Stimulation of rabbit synovial fibroblasts with 10 ng/ml recombinant human IL-1 beta resulted in a 20-fold increase in collagenase mRNA by 12 h. Transient transfection studies using collagenase promoter-CAT constructs demonstrated that proximal sequences responded poorly to IL-1 beta, possibly due to insufficient activation of AP-1 by this cytokine. More distal sequences were required for IL-1 beta responsiveness, with a 4700 bp construct showing approximately 5-fold induction above control. To examine post-transcriptional mechanisms, transcript from a human collagenase cDNA was constitutively produced by the simian virus 40 early promoter. IL-1 beta stabilized the constitutively expressed human transcript. Furthermore, mutation of the ATTTA motifs in the 3' untranslated region of the human gene also stabilized the transcript. Finally, the rabbit collagenase 3' untranslated region destabilized a constitutively transcribed chloramphenicol acetyltransferase transcript. These data indicate that in addition to activating transcription, IL-1 beta increases collagenase transcript stability by reversing the destabilizing effects of sequences in the 3' untranslated region.

Homeodomain protein binding sites, inverted repeats, and nuclear matrix attachment regions along the human beta-globin gene complex.

beta-Globin genes in primates arose during evolution by duplication of an ancestral gene, and their order of arrangement along the DNA is related to their timing of expression during development. We believe that nuclear matrix anchorage sites (MARs) along the beta-globin gene complex considered to be mass binding sites for transcription protein factors, some of which are developmental stage specific and others ubiquitous, play a decisive role in cell memory by determining the developmental stage-specific expression of the genes. The AT-rich class of MARs appears to possess a significant number of ATTA and ATTTA motifs known to be mass binding sites for homeodomain proteins that determine body formation in development. MARs also appear to harbor origins of replication, to be enriched in inverted repeats (dyad symmetry motifs) and were proposed to include the DNase I hypersensitive sites of a particular gene determined at the chromatin level. This study is an attempt to finely identify MARs at the nucleotide level along the beta-globin gene complex. Searches of a contiguous stretch of about 73.3 kb of human sequences comprising and surrounding the epsilon, gamma G-, gamma A-, delta-, and beta-globin genes of the human beta-globin gene complex for homeotic protein binding sites as well as for inverted repeats has shown that these elements are clustered nonrandomly at particular sites within the beta-globin gene complex. These sites are presumed to be the AT-rich class of MARs of the beta-globin gene complex. The inverted repeats which are characteristic of origins of replication and some promoter/enhancer regions and the homeotic protein sites seem to include the DNase I hypersensitive sites of the gene complex. Indeed, dyad symmetry sequences are present close to the four DNase I HS sites in the locus control region (LCR) of the gene complex as well as in the 5' flanking regions and the large introns of the delta- and beta-globin genes. A search of the putative MAR regions of the gene complex suggests that, in addition to their enrichment in ATTA motifs, palindromes, and DNase I hypersensitive sites, these regions may comprise TG-rich motifs and potential Z-DNA as well as polypurine and polypyrimidine blocks. From the positions of palindromes and clusters of homeodomain protein sites along the complex we propose that an extended origin of replication able to initiate at several sites is present in the LCR and two others surrounding the delta- and beta-globin genes.(ABSTRACT TRUNCATED AT 400 WORDS)