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

Previous reports suggest that type I and type II Interferon can co-operatively inhibit some virus replication, e.g. HCV, SARS-CoV, HSV-1. To find out the molecular mechanism underlying this phenomenon, we analyzed the transcription profile stimulated by IFN-α and IFN-γ in Huh-7 cells and found that the transcription of a subset of IFN stimulated genes (ISGs) including BclG, XAF1, TRAIL and TAP1 was enhanced when IFN-α and γ were both present. Promoter analysis of BclG revealed that IRF-1 and STAT1 were both required in this process. Enhanced IRF-1/DNA complex formation was observed in interferon co-treatment group by gel shift analysis. Furthermore, IRF-1 activation was found to be generally required in this cluster of ISGs. STAT1 tyrosine phosphorylation was elevated by IFN combination treatment, however, only the hyper-transactivation of GAS but not ISRE was observed. In conclusion, hyper-activation of IRF-1 and elevated STAT1 dimer formation may be two general switches which contribute to a much more robust antiviral symphony against virus replication when type I and type II IFNs are co-administered.

The role of two genes, mlcR and ariB, was investigated by gene disruption experiments. The mlcR gene in the ML-236B biosynthetic gene cluster of Penicillium citrinum encodes a putative 50.2-kDa protein with a Zn (II) ₂Cys₆ DNA-binding domain, and has similarity to most of the GAL4-type regulatory proteins. The mlcR disruptant did not produce ML-236B or its intermediates, suggesting that mlcR is involved in ML-236B biosynthesis. Transcriptional analysis of the mlcR disruptant by Northern hybridization and RT-PCR indicated that MlcR activates the transcription of mlcA, B, C,D, F, G and H in a pathway-specific manner. On the other hand, MlcR did not affect the transcription of mlcE and the genes outside the ML-236B cluster. The ariB gene, next to mlcR, encodes another GAL4-type protein. Transcriptional analysis of the ariB disruptant indicated that it is a transcriptional activator of the genes outside the ML-236B cluster, and is not related to ML-236B biosynthesis.

Mutations in an ABC transporter gene called ABCC6 are responsible for pseudoxanthoma elasticum (PXE), a rare heritable disease characterized by elastic fiber calcification in skin, ocular and vascular tissues. The presumed function of this ABC transporter is to export metabolites from polarized cells. However, the endogenous substrate(s) are unknown and the exact relationship with elastic fibers is unclear. As ABCC6 is only expressed at high level in liver and kidneys, tissues seemingly unrelated to the PXE phenotype, we explored the transcriptional regulation of the murine Abcc6 gene to define the transcriptional signal conferring tissue specificity and to gather clues on its possible biological function. We cloned 2.9 kb of the mAbcc6 5′-flanking region and several deletion constructs linked to a luciferase reporter gene. We delineated a proximal promoter and a liver-specific enhancer region. We also demonstrated that the proximal region is a TATA-less promoter requiring an intact CCAAT-box and Sp1 binding for its basal activity. By using reporter assays and chromatin immunoprecipitations, we showed that HNF4α and surprisingly, NF-E2, enhanced the mAbcc6 promoter activity. The involvement of both HNF4α and NF-E2 in the mAbcc6 gene regulation suggests that Abcc6 might be involved in a detoxification processes related to hemoglobin or heme.

The kdp region from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius consists of two divergent operons: kdpZFABCN, which is tenfold induced at low K⁺ concentrations and encodes the K⁺-translocating P-type ATPase KdpZFABC as well as KdpN, a novel covalent homo-dimer of the cytoplasmic N-terminal part from sensor kinase KdpD; and secondly, the constitutively expressed kdpHE operon, encoding the remainder of KdpD and the response regulator KdpE.

Initiation of RNA transcription may be a rate-limiting step in gene expression but it is only the first of many regulatory processes that impinge on nascent RNA along its path to maturity. Discontinuity between gene expression patterns within the nucleus and the cytoplasm suggests that multiple post-transcription regulatory points greatly influence the final RNA product, even to the extent of dramatically shifting the gene targets identified as a defined regulatory response.

We have cloned a porcine orthologue of cofactor CLIM2 (Ldb1/NLI) from the porcine pituitary cDNA library by protein–protein interaction with the Yeast Two-Hybrid System using porcine Lhx2 as a bait protein. Porcine CLIM2 shows a high identity (99%) in the dimerization domain, nuclear localization signal and LIM binding domain with those of man and mouse. The expression of CLIM2 gene in the anterior pituitary lobe was detected during the porcine fetal and postnatal period by RT-PCR analysis, suggesting that this protein is constitutively expressing and plays a basic role in the anterior pituitary. Transfection assay to the pituitary tumor derived LβT2 cells, and the Chinese hamster ovary cells demonstrated that CLIM2 acts as a corepressor of the porcine Lhx2 function. Interestingly, CLIM2 alone apparently repressed the high level of αGSU gene expression in LβT2 cells. These data suggest that CLIM2 is a basic factor in the pituitary development and function, and plays the role of repressor to modify the function of Lhx2 on the αGSU gene expression.

The human amyloid precursor protein (APP) is the precursor of Aβ, a peptide with the potential to create amyloid plaques in neurons. Mutations in the human APP gene are associated with the familial form of Alzheimer's disease. In addition, differential expression of three alternative pre-mRNA APP splicing variants of 695, 751, and 770 amino acids is linked to the pathogenesis. In this study, two novel transcript variants of porcine APP have been identified, producing isoforms of 695 and 751 amino acids, respectively. These are highly homologous to APP orthologues from other vertebrate species. Expression analyses revealed that the gene is expressed in all 30 examined porcine tissues and in a selected subset of these, differential representation of the three major APP transcript variants was observed. The APP isoform of 770 amino acids clearly predominates in non-neuronal tissues while in porcine cerebellum, the APP isoforms of 695 and 770 amino acids are expressed at equivalent levels. Employing a somatic cell hybrid panel, the APP gene was mapped to porcine chromosome 13 in either the 13q41 or 13q46–q49 region. A large pig population was screened for single nucleotide polymorphisms (SNPs) in APP exon 17 and flanking intron sequences. No missense mutations were detected; however, the allele frequencies of two silent mutations and two intron polymorphisms varied significantly among breeds.

Serotonin (5-HT) mediates a number of diverse physiological functions in crustaceans by interacting with various 5-HT receptor subtypes. A putative 5-HT receptor cloned from the ovary of the black tiger prawn (Penaeus monodon) consisted of 2291 nucleotides, encoding a putative 5-HT_1Pem receptor protein of 591 amino acids. Transient expression of 5-HT_1Pem in HEK293 cells demonstrated a saturable [³H]-5-HT binding with a Kd of 10.43 ± 1.13 nM and B_max of 1.53 ± 0.06 pmol/mg. The putative 5-HT_1Pem receptor is expressed in all tissues examined and is constitutively expressed in the ovary during ovarian maturation and spent phase. Polyclonal antibodies against the third intracellular loop (i3 loop) of the 5-HT receptor showed that the 5-HT_1Pem receptor protein was expressed in the trabeculae of ovarian stages 1 and 2 but on the cortical rod and surrounding the oocyte membrane of stages 3 and 4, suggesting that receptor localization plays a critical role in regulating ovarian maturation and spawning in penaeus shrimp.

Mechanisms to maintain blood pressure in the face of infection are critical to survival. The angiotensinogen (AGT) gene locus is an important component of this response. Thus the AGT gene, expressed predominantly by liver cells, is known to be a positive acute phase reactant. We have previously demonstrated activation of the AGT promoter in hepatocytes through the IL6/STAT3 signaling mechanism. We have now investigated whether IFN-gamma, a cytokine also induced in response to diverse infections, can regulate AGT gene expression, and have elucidated the molecular mechanism involved. IFN gamma treatment up-regulated AGT mRNA level and promoter activity in Hep3B hepatocytes. Sequential deletion of the promoter from the 5′ side suggested the major IFN gamma responsive DNA element to be between − 303 and − 103. This region contained a candidate STAT1-binding site between − 271 and − 279. EMSA and chromatin immuno-precipitation (ChIP) assays confirmed that IFN-gamma treatment induced the binding of STAT1 to this element. Reporter constructs containing this AGT promoter derived element in a multimerized context but not a mutant version were responsive to IFN gamma. Moreover mutating this STAT1 element in the context of the wild-type AGT holo promoter reduced responsiveness to IFN gamma. In contrast to the clear synergism between dexamethasone and IL 6 in the upregulation of the AGT promoter (through interaction between GR and STAT3), the combination of IFN gamma with IL 6 or with dexamethasone did not further increase AGT promoter activity suggesting that the IFN gamma/STAT1 pathway represents a separate signaling mechanism. These data highlight the redundancy in cytokine-mediated host response pathways aimed at the maintenance of blood pressure during infection.

All human GM₃ synthase mRNA variants until now identified predict a protein of 362 amino acids having substrate activity highly restricted to lactosylceramide. In this report we describe the identification of a new GM₃ synthase transcript containing an additional translation start codon, located upstream and in-frame with that up to now considered unique translation initiation site in the human GM₃ synthase gene. In vitro expression studies showed that the new transcript produces a longer form of human GM₃ synthase, that is efficiently translocated into the microsomal lumen and glycosylated. Moreover, stable cDNA transfection into mammalian cells gives rise to a threefold increase of GM₃ synthase activity, associated to a broader substrate specificity. Although this transcript has been initially identified in the human placenta, RT-PCR analyses verified the expression of an identical mRNA also in undifferentiated HL60 cells, but not in the monocytic lineage. Altogether, these results are the first demonstration of the existence of a new isoform of human GM₃ synthase, which could play an important role during HL60 cell differentiation. The functional relevance of the existence of two isoforms of GM₃ synthase is also discussed.