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

Ser/Thr- and Tyr-Protein kinases constitute a key switch underlying the dynamic nature and graded regulation of signal transduction and pathway activities in cellular organization. Here we describe the identification and characterization of Dusty, a single-copy gene that arose in metazoan evolution and encodes a putative dual Ser/Thr and Tyr protein kinase with unique structural features. Dusty is widely expressed in vertebrates, broadly distributed in the central nervous system, and deregulated in certain human cancers. Confocal imaging of transiently expressed human Dusty-GFP fusion proteins showed a cytoplasmic distribution. Dusty proteins from lower to higher species display an increasing degree of sequence conservation from the N-terminal non-catalytic domain to C-terminal catalytic domain. The non-catalytic region has eight conserved cysteine residues, multiple potential kinase-docking motifs and phosphorylation sites, whereas the catalytic domain is divergent and about equally distant of Ser/Thr and Tyr protein kinases. Homology analyses identified the essential catalytic residues, suggesting that Dusty homologues all possess the enzymatic activity of a protein kinase. Taken together, Dusty is a unique evolutionarily selected group of divergent protein kinases that may play important functional roles in the brain and other tissues of vertebrates.

Alfalfa leaf protoplast-derived cells can develop into somatic embryos depending on the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the initial culture medium. In order to reveal gene expression changes during the establishment of embryogenic competence, we compared the cell types developed in the presence of 1 and 10 μM 2,4-D, respectively, at the time of their first cell divisions (fourth day of culture) using a PCR-based cDNA subtraction approach. Although the subtraction efficiency was relatively low, applying an additional differential screening step allowed the identification of 38 10 μM 2,4-D up-regulated transcripts. The corresponding genes/proteins were annotated and representatives of various functional groups were selected for more detailed gene expression analysis. Real-time quantitative PCR (RT-QPCR) analysis was used to determine relative expression of the selected genes in 2,4-D-treated leaves as well as during the whole process of somatic embryogenesis. Gene expression patterns confirmed 2,4-D inducibility for all but one of the 11 investigated genes as well as for the positive control leafy cotyledon1 (MsLEC1) gene. The characterized genes exhibited differential expression patterns during the early induction phase and the late embryo differentiation phase of somatic embryogenesis. Genes coding for a GST-transferase, a PR10 pathogenesis-related protein, a cell division-related ribosomal (S3a) protein, an ARF-type small GTPase and the nucleosome assembly factor family SET protein exhibited higher relative expression not only during the induction of somatic embryogenesis but at the time of somatic embryo differentiation as well. This may indicate that the expression of these genes is associated with developmental transitions (differentiation as well as de-differentiation) during the process of somatic embryogenesis.

The HKα2 gene directs synthesis of the HKα2 subunit of the H⁺, K⁺-ATPase. In the kidney and colon, the gene is highly expressed and is thought to play a role in potassium (K⁺) conservation. The rabbit has been an important experimental system for physiological studies of ion transport in the kidney, so the rabbit HKα2 gene has been cloned and characterized. The genomic clones and the previously reported HKα2a and HKα2c subunit cDNAs provided a means to address several issues regarding the structure and expression of the HKα2 gene. First, the genomic organization established that the rabbit HKα2 gene was unambiguously homologous to the mouse HKα2 gene and the human ATP1AL1 gene. Second, the mapping of the transcription start site for the alternate transcript, HKα2c, confirmed that it was an authentic rabbit transcript. Finally, isolation of DNA from the 5′ end of the HKα2 gene enabled us to initiate studies on its regulation in the rabbit cortical collecting duct. The promoter and two putative negative regulatory regions were identified and the effect of cell confluency on gene expression was studied.

Activation of transcription by NF-κB requires association with coactivator proteins, including CBP/p300 and P/CAF. To identify new coregulatory proteins, a cytoplasmic two-hybrid screen was performed using the C-terminus of the p65 subunit as bait. Through this screen, the spermidine/spermine N¹-acetyltransferase 2 (SSAT2) protein was identified as a potential modulator of NF-κB activity. SSAT2 was originally identified based on homology to SSAT1, a protein involved in polyamine catabolism. However both proteins contain an acetyltransferase domain that has similarity to the acetyltransferase domains of the GNAT superfamily of coactivators. Although SSAT2 is 46% identical to SSAT1, based on a recent report, SSAT2 does not appear to function in polyamine catabolism. Because of the similarity of SSAT2 to coactivators, we wanted to determine if SSAT2 could function as a coactivator for NF-κB. Coimmunoprecipitations confirmed the interaction between p65 and SSAT2. In transient transfection reporter gene assays, SSAT2 functions as a transcriptional coactivator for NF-κB and cooperates with CBP and P/CAF to enhance TNFα-induced NF-κB activity. Moreover, SSAT2 transiently associates with the promoters of the NF-κB-regulated cIAP2 and IL-8 genes in response to TNFα. Although the overall function of SSAT2 is not known, it appears that it can function as a transcriptional coactivator.

The growth arrest and DNA damage-inducible gene 45A (GADD45A) is involved in the DNA repair, maintenance of genomic stability, cell cycle control and apoptosis, and thus plays an important role in cellular response to DNA damage. The GADD45A gene is responsive to a variety of DNA-damaging agents, including ionizing radiation (IR), methyl methanesulfonate (MMS), and ultraviolet (UV) radiation. It is generally thought that induction of the GADD45A gene after IR exposure is principally p53-dependent, requiring binding of the p53 protein to the p53-recognition sequence in the third intron. However, the involvement of factors other than p53 in transcriptional regulation of the GADD45A gene after IR exposure has not been elucidated. In the present study, we show that the 5′-flanking region containing two OCT sites and a CCAAT box, as well as p53 and AP-1 sites in the third intron, are required for the basal transcriptional activity of the reporter gene. In addition, AP-1 recognition element was shown to be involved in the transcriptional enhancement of the GADD45A gene after X-ray irradiation. Electrophoretic mobility shift analysis (EMSA) and Chromatin immunoprecipitation (ChIP) assay revealed that JunD binds to the third intron of the GADD45A gene. These observations suggest that AP-1 complexes containing JunD, in addition to p53, play an important role not only in transcriptional enhancement by IR but also in basal expression of the GADD45A gene via binding to the AP-1 site in the third intron.

During germination of cereal seeds, aleurone cells respond to gibberellins (GA) by synthesizing and secreting hydrolytic enzymes that mobilize the reserved nutrients. It has been shown that products of early GA response genes, like a transcription factor GAMyb, act as key molecules leading to this regulation. Pivotal roles of GAMyb on expression of hydrolase genes have been well documented, whereas regulation of GAMyb expression itself remains obscure. In order to understand virtual mechanisms of the GA-mediated expression of genes, it is important to know how GA control expression of early GA response genes. Using an aleurone transient expression system of rice (Oryza sativa L.), we examined GA responsive domains of early GA response genes in the aleurone, such as GAMyb and OsDof3. The upstream promoter could not confer GA response. Extensive analyses revealed the presence of enhancer-like activities in a large first intron. In Arabidopsis, intron enhancers have been identified in MADS-box homeotic genes, AGAMOUS (AG) and FLOWERING LOCUS C (FLC), in which large introns should not only confer proper gene expressions, but also associate with gene silencing by covalent modifications of both DNA and histone. These evidences prompt us to assign that chromatin-based control might be important for initial GA action. Based on this assumption, we have identified DNA methylation of the GAMyb locus in germinated rice seeds.

The Trypanosoma cruzi rRNA gene promoter was characterized by deletion and point mutation analyses. A core of 89 bp was identified as the minimal region with full promoter activity. This core region is flanked upstream by a control element that stimulates its activity, and downstream by a novel down regulating region of about 200 bp. A point mutation analysis of the transcription start region evidenced 7 contiguous nucleotides where individual substitutions produced in all cases a defective promoter. It is generally accepted that the anciently speciated trypanosomatids lack strict promoters for protein coding genes transcribed by RNA polymerase II. The occurrence of a well structured rRNA gene promoter in these species suggests an early appearance of the RNA polymerase I promoters in the evolution of eukaryotic cells.

Expression of the tissue-specific gap junction protein connexin(Cx)40 is regulated by the interaction of ubiquitous and tissue-specific factors such as Sp1 and GATA4. Cardiac Cx40 expression is altered under pathological conditions such as atrial fibrillation. A human promoter polymorphism, a G → A change at position − 44 that has been associated with atrial-specific arrhythmias, is located between the TBE-NKE-Sp and GATA consensus transcription factor binding sites important for the regulation of the mouse Cx40 gene. The presence of the A-allele at position − 44 in promoter–reporter constructs significantly reduces promoter activity. Using electrophoretic mobility shift assays and luciferase reporter assays in various cell types, we show that Sp1 and GATA4 are important regulators of human Cx40 gene transcription and that the − 44 G → A polymorphism negatively affects the promoter regulation by the transcription factors Sp1 and GATA4.

In this study, we have shown the transcriptional regulation of the human Sia-alpha2,3-Gal-beta1,4-GlcNAc-R:alpha2,8-sialyltransferase (hST8Sia III) induced by retinoic acid (RA), a potent neuronal cell regulator in glioblastoma cell line (U-87MG). The induction of hST8Sia III by RA is regulated at the transcriptional level in a dose- and time-dependent manner, as evidenced by reverse transcription-polymerase chain reaction (RT-PCR). To elucidate the mechanism underlying the regulation of hST8Sia III gene expression in RA-stimulated U-87MG cells, we characterized the promoter region of the hST8Sia III gene. Functional analysis of the 5′-flanking region of the hST8Sia III gene by the transient expression method showed that the − 1194 to − 816 region, which contains a retinoic acid nucleic receptor (RAR) at − 1000 to − 982, functions as the RA-inducible promoter in U-87MG cells. Site-directed mutagenesis indicated that the RA binding site at − 996 to − 991 is crucial for the RA-induced expression of the hST8Sia III in U-87MG cells. In addition, the transcriptional activity of hST8Sia III induced by RA in U-87MG cells was strongly inhibited by SP600125, c-Jun N-terminal Kinase (JNK) inhibitor, as determined by RT-PCR and luciferase assay of hST8Sia III promoter containing the − 1194 to − 816 regions. These results suggest that RA markedly modulates transcriptional regulation of hST8Sia III gene expression through JNK signal pathway in U-87MG cells.

The aryl hydrocarbon receptor (AHR) mounts the body's main molecular defense against environmental toxicants by inducing a battery of genes encoding xenobiotic metabolizing proteins. The AHR is activated by polycyclic aromatic hydrocarbon toxicants, including the pervasive teratogen and carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). The TCDD-activated AHR significantly changes the cytoplasmic mRNA levels of hundreds of genes, but little is known of the mechanism by which the activated AHR causes such a strong effect on global gene expression. We used high-density microarrays to compare nuclear and cytoplasmic RNA levels from untreated and TCDD-treated mouse embryonic fibroblasts (MEF) to test the hypotheses that (1) TCDD has a large impact on nuclear RNA levels and (2) that cytoplasmic RNA levels are dependent on nuclear RNA levels. We found that nuclear RNA levels are strongly affected by TCDD, and that nuclear and cytoplasmic RNA levels are only weakly correlated, indicating that other regulatory mechanisms are controlling cytoplasmic RNA levels. The nuclear RNAs most affected by TCDD encode proteins involved in nuclear RNA processing and transcription. We conclude that although the AHR regulates key xenobiotic metabolizing genes at the transcriptional level, a larger impact of the TCDD-activated AHR may be at post-transcriptional levels.