Genomic organization of the gene that encodes the precursor to EGF-related peptides, exogastrula-inducing peptides, of the sea urchin Anthocidaris crassispina

Exogastrula-inducing peptides (EGIPs) were identified in embryos of the sea urchin Anthocidaris crassispina as polypeptides with structural similarity to epidermal growth factor (EGF) that severely affect gastrulation of sea urchin embryos to induce exogastrulation. Here we have obtained genomic clones for the EGIP precursor gene (EGIP) and determined its genomic organization. The EGIP gene spans the length of 9 kb in the genome and is composed of seven exons and six introns. Each of the four EGF motifs in the precursor protein is encoded by a single exon, and all the exon boundaries are in phase 1, suggesting that EGIP have been generated during evolution by duplication of an exon encoding a single ancient EGIP sequence. The 5'-flanking sequence of EGIP from -4372 to +194 revealed the presence of multiple repeat sequences including direct and inverted repeats as well as two clusters of GGGG/CCCC elements. The function of the upstream flanking region of EGIP was examined by introducing the gene constructs into embryos in which different regions from the flanking DNA were placed upstream to the GFP reporter gene. Systematic deletion of the upstream DNA revealed the presence of potent enhancer activity between -372 and -210.     

非洲爪蟾肌肉决定基因Xmyf—5表达调控的研究

Ｘｍｙｆ－５是爪蟾胚胎肌细胞决定的关键基因之一,研究Ｘｍｙｆ－５的表达调控有助于揭示肌肉原基的形成和肌肉发育的分子机制。从爪蟾部分基因组文库筛选到Ｘｍｙｆ－５５‘上游４．９ｋｂ片段。该片段指导报告基因在爪蟾胚胎内的表达以及其缺失片段指导的报告基因活性分析结果显示,Ｘｍ６ｓｆ－５５’上游４．９ｋｂ片段内含有指导Ｘｎｙｆ－５在胚胎内的表达以及其缺失片段指导的报告基因活性分析结果显示,Ｘｍｙｆ－５５‘     

Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice.

Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms.     

Complete nucleotide sequence of the chicken alpha A-crystallin gene and its 5' flanking region

The chicken alpha A-crystallin gene and 2.6 kb of its 5' flanking sequence have been isolated and characterized by electron microscopy and sequencing. The structural gene is 4.5 kb long and contains two introns, each approx. 1 kb in length. The first intron divides codons 63 and 64, and the second intron divides codons 104 and 105, as in rodents. There is little indication that the insert exon of rodents (an alternatively spliced sequence) is present in complete form in the chicken alpha A-crystallin gene; small stretches of similarity to this sequence were found throughout the gene. The 5' flanking sequence of the chicken alpha A-crystallin gene shows considerable sequence similarity with other mammalian alpha B-crystallin genes. In addition, one consensus sequence (GCAGCATGCCCTCCTAG) present in the 5' flanking region of the chicken alpha A-crystallin gene was found in the 5' flanking region of most reported crystallin genes.     

The human ubiquitous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene (PFKFB3): promoter characterization and genomic structure

A DNA fragment containing 1.5 kb of the 5'-flanking region of the human ubiquitous PFKFB3 gene, coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was cloned and its promoter activity was examined. The 5' flanking region contains a TATA box-like and GC-rich sequences, yielding several potential Specific protein (Sp-1) and activator protein (AP)-2 binding sites. Putative regulatory motifs for E-box, nuclear factor (NF)-1 and progesterone response element were also found by computer assisted analysis. Transient expression assays of truncated promoter-reporter constructs in HeLa cells showed that this gene is induced by phorbol esters (PDB) and cyclic-AMP-dependent protein kinase signal activation. Furthermore, the genomic organization of the PFKFB3 gene is reported. This gene spans more than 26 kb containing at least 16 exons that accounts for the two reported isoforms, inducible and ubiquitous, generated through alternative splicing of exon 15.     

A GATA box in the GATA-1 gene hematopoietic enhancer is a critical element in the network of GATA factors and sites that regulate this gene

A region located at kbp -3.9 to -2.6 5' to the first hematopoietic exon of the GATA-1 gene is necessary to recapitulate gene expression in both the primitive and definitive erythroid lineages. In transfection analyses, this region activated reporter gene expression from an artificial promoter in a position- and orientation-independent manner, indicating that the region functions as the GATA-1 gene hematopoietic enhancer (G1HE). However, when analyzed in transgenic embryos in vivo, G1HE activity was orientation dependent and also required the presence of the endogenous GATA-1 gene hematopoietic promoter. To define the boundaries of G1HE, a series of deletion constructs were prepared and tested in transfection and transgenic mice analyses. We show that G1HE contains a 149-bp core region which is critical for GATA-1 gene expression in both primitive and definitive erythroid cells but that expression in megakaryocytes requires the core plus additional sequences from G1HE. This core region contains one GATA, one GAT, and two E boxes. Mutational analyses revealed that only the GATA box is critical for gene-regulatory activity. Importantly, G1HE was active in SCL(-/-) embryos. These results thus demonstrate the presence of a critical network of GATA factors and GATA binding sites that controls the expression of this gene.     

Genomic organization and the 5' upstream sequences associated with the specific spatio-temporal expression of HrEpiC, an epidermis-specific gene of the ascidian Halocynthia roretzi.

An epidermis-specific gene HrEpiC of the ascidian Halocynthia roretzi is activated in all presumptive blastomeres by the 64-cell stage. To explore the molecular mechanisms underlying the regulation of the timing of activation of HrEpiC, we studied the genomic organization and the 5' upstream sequences of HrEpiC associated with specific spatio-temporal expression of the gene. The restriction site mapping and sequencing of genomic clones showed that the H. roretzi genome contained two copies of HrEpiC gene, HrEpiC1 and HrEpiC2, aligned tandemly in about 8 kb of the genome. Analysis of various deletion constructs with the 5' flanking sequences of HrEpiC1 revealed that 103 bp of the 5' flanking region was sufficient for the minimal epidermis-specific expression of HrEpiC1 and that the region between -281 bp and -198 bp of the 5' flanking region was associated with the amplification of the minimal expression of the reporter gene in the epidermis. This module between -281 bp and -198 bp was also shown to be associated with the timing of the activation of HrEpiC1 by the 64-cell stage. We discussed how the spatio-temporal expression pattern of HrEpiC1 is regulated by the two modules.     

Identification of New Regulatory Sequences Far Upstream of the Mouse Monocyte Chemoattractant Protein-1 Gene

We systematically searched for sequences influencing the expression of the mouse monocyte chemoattractant protein-1 (MCP-1) gene (Scya2) by mapping DNase I hypersensitive sites (HS) in the chromatin of mesangial cells in a 40-kb interval around the gene. We found nine HS located between -24 kb and +12.7 kb. Three HS coincided with previously known regulatory sequences (HS-2.4, HS-1.0, and HS-0.2). We tested two of the previously unknown HS located far upstream of Scya2 (HS-19.4 and HS-16.3) in transfection experiments using luciferase reporter constructs and mouse mesangial cells as recipients. In transient transfections, both HS had a moderate effect on basal promoter activity as well as promoter activity stimulated by tumor necrosis factor-alpha. In stable transfection experiments, we found much higher activity. A DNA fragment containing HS-19.4 and HS-16.3 caused a considerable increase in the number of stably integrated luciferase copies. We determined the nucleotide sequence of the 5' flanking region to -28.6 kb. Computer-assisted sequence analysis did not yield evidence of an additional gene. These HS are located within the 5' flanking region of a gene cluster consisting of Scya2 (MCP-1), Scya7 (MCP-3), Scya11 (eotaxin), Scya12 (MCP-5), and Scya8 (MCP-2). This report represents the first comprehensive chromatin analysis of the mouse MCP-1 locus leading to the identification of a complex regulatory region located far upstream of Scya2.     

Vasopressin-SV40 T antigen expression in transgenic mice induces brain tumor and lymphoma

In order to understand the importance of various cis-acting elements in regulating VP gene expression, transgenic mice regulated by VP constructs were produced containing 3.8 kb of the 5' flanking region and all the exons and introns in the mouse VP gene, which was fused at the end of exon 3 to an SV40 T antigen (Tag). In the transgenic mice by the pVPSV.IGR3.6 construct, all the six transgenic mice died at the age of 2-6 weeks. In the transgenic mice by pVPSV.IGR2.1, 21% of them had brain tumors at 5 weeks and 100% of the mice had brain tumors after 24 weeks. Histological analysis of the transgenic mice revealed primitive neuroectodermal tumors (PNET) in the brain and lymphoma in the spleen and lymph nodes. The phenotype differences between the two transgenic mice suggest that tissue-specific expression might be regulated by cis-acting elements in the 1.5-kb of the 3(') flanking region, which are not contained in pVPSV.IGR2.1. In conclusion, pVPSV.IGR2.1 mice will be a valuable mouse model system for investigating PNET tumorigenesis in the brain and lymphoma in the lymph nodes and spleen.     

Isolation and characterisation of tilapia beta-actin promoter and comparison of its activity with carp beta-actin promoter

The regulatory sequence including proximal promoter, untranslated exon 1 and intron 1 of the beta-actin gene from tilapia (Oreochromis niloticus) has been isolated and spliced to a beta-galactosidase reporter gene to test its activity. Comparisons of promoter activity have been carried out with three different constructs: (1) 1.6 kb tilapia beta-actin regulatory sequence, (2) 1.5 kb carp beta-actin regulatory sequence, and (3) 4.7 kb carp beta-actin regulatory sequence. Although the 1.6 kb tilapia beta-actin regulatory sequence gave slightly different expression patterns in tilapia embryos assayed by in situ X-gal staining, no difference was observed in expression level when the tilapia sequence was compared with the 4.7 kb carp beta-actin regulatory sequence by quantitative assay. In comparison with the 1.5 kb carp beta-actin regulatory sequence, the 1.6 kb tilapia beta-actin regulatory sequence gave higher expression levels in tilapia embryos, while a reverse result was observed in zebrafish embryos. In cell transfection experiments, the 1.6 kb tilapia beta-actin regulatory sequence showed three to four times better activity in blue gill cells than either the 4.7 kb carp beta-actin or the 1.5 kb carp beta-actin regulatory sequences. The 1.6 kb tilapia beta-actin regulatory sequence also drove higher reporter gene activity in somatic cells of tilapia than did the 4.7 kb carp beta-actin regulatory sequence following direct injection of constructs into muscle. Therefore, taken together, the data demonstrate that the tilapia beta-actin promoter can be used as an efficient regulatory sequence to produce autotransgenic tilapia.