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BBA - Gene Structure and Expression (v.1759, #7)

Editorial Board (pp. ii).

Biological microarray interpretation: The rules of engagement by Rainer Breitling (pp. 319-327).

Gene expression microarrays are now established as a standard tool in biological and biochemical laboratories. Interpreting the masses of data generated by this technology poses a number of unusual new challenges. Over the past few years a consensus has begun to emerge concerning the most important pitfalls and the proper ways to avoid them. This review provides an overview of these ideas, beginning with relevant aspects of experimental design and normalization, but focusing in particular on the various tools and concepts that help to interpret microarray results. These new approaches make it much easier to extract biologically relevant and reliable hypotheses in an objective and reasonably unbiased fashion.

Keywords: Microarray; Gene expression; Bioinformatics; Tool; Computational biology; System biology

Molecular cloning and functional expression of the Penaeus monodon 5-HT receptor by Chalermporn Ongvarrasopone; Yaowaluck Roshorm; Suthasinee Somyong; Chetsada Pothiratana; Soontaree Petchdee; Jarasporn Tangkhabuanbutra; Samaisukh Sophasan; Sakol Panyim (pp. 328-339).

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.

Keywords: Serotonin receptor; G-protein coupled receptor; Black tiger shrimp; Ovarian maturation; Immunohistochemistry

Upregulation of human angiotensinogen (AGT) gene transcription by interferon–gamma: Involvement of the STAT1-binding motif in the AGT promoter by Sudhir Jain; Mehul Shah; Yanna Li; Govindaiah Vinukonda; Pravin B. Sehgal; Ashok Kumar (pp. 340-347).

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.

Keywords: Cytokine; Transcription factor; Gene regulation; Inflammation

Human GM₃ synthase: a new mRNA variant encodes an NH₂-terminal extended form of the protein by Patrizia Berselli; Stefania Zava; Elena Sottocornola; Simona Milani; Bruno Berra; Irma Colombo (pp. 348-358).

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.

Keywords: GM; ₃; synthase; GM; ₃; Transcription; In vitro translation; Human placenta; HL60 cell differentiation

Drosophila distal-less negatively regulates dDREF by inhibiting its DNA binding activity by Yuko Hayashi; Masaki Kato; Hirokazu Seto; Masamitsu Yamaguchi (pp. 359-366).

The Drosophila DNA replication-related element binding factor (dDREF) is required for expression of many proliferation-related genes carrying the DRE sequence, 5′-TATCGATA. Over-expression of dDREF in the eye imaginal disc induces ectopic DNA synthesis, apoptosis and inhibition of photoreceptor cell specification, and results in rough eye phenotype in adults. In the present study, half dose reduction of the Distal-less ( Dll) gene enhanced the dDREF-induced rough eye phenotype, suggesting that Dll negatively regulates dDREF activity in eye imaginal disc cells. Biochemical analyses revealed the N-terminal (30aa to 124aa) and C-terminal (190aa to 327aa) regions of Dll to interact with the DNA binding domain (16aa to 125aa) of dDREF, although it is not clear yet whether the interaction is direct or indirect. Electrophoretic mobility shift assays showed that Dll thereby inhibits DNA binding. The repression of this dDREF-function by a homeodomain protein like Dll may contribute to the differentiation-coupled repression of cell proliferation during development.

Keywords: DNA replication-related element binding factor (DREF); Distal-less; DNA binding; Proliferation; Transcription factor

The characterisation of the human Wolfram syndrome gene promoter demonstrating regulation by Sp1 and Sp3 transcription factors by Christopher Ricketts; Malgorzata Zatyka; Timothy Barrett (pp. 367-377).

Wolfram Syndrome (DIDMOAD) is an autosomal recessive disorder characterised by insulin deficient diabetes mellitus and neurodegeneration. Mutations in a novel gene, WFS1, were found in nearly all patients and segregated with the disease. The WFS1 gene is expressed in all tissue types studied and the 890aa protein product is localised to the endoplasmic reticulum (ER). In this study, we used a combination of reporter assays and in vitro and in vivo transcription factor binding assays to analyse the regulation of expression of the human WFS1 gene in neuronal derived cells. A single transcription start site was mapped and a minimal promoter identified within 25 bp upstream of this site. This minimal promoter contains two DNA binding motifs (GC boxes) for the transcription factors Sp1/3/4 and binding of both Sp1 and Sp3 was demonstrated at both motifs in vitro and in vivo. The presence of intact GC boxes is essential for minimal promoter action. Thus, transcription factors of the Sp family are important regulators of the WFS1 promoter. A further up-regulating control region was identified containing three CCAAT box binding motifs; all demonstrated a reduction in expression after mutation. One CCAAT box represented part of a predicted ER stress response element.

Keywords: WFS1; Wolfram syndrome; Minimal promoter; Sp1; Sp3; CCAAT; ERSE

Porcine APP cDNAs: Molecular cloning and characterization, expression analysis, chromosomal localization and SNP analysis by Marianne Abildgaard Oerum; Christian Bendixen; Lone Bruhn Madsen; Knud Larsen (pp. 378-384).

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.

Keywords: Alzheimer's disease; Amyloid precursor protein; Comparative mapping; Expression analysis; Polymorphism; Sus scrofa

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BBA - Gene Structure and Expression (v.1759, #7)