Specific expression in mouse mesoderm- and neural crest-derived tissues of a human PDGFRA promoter/lacZ transgene

The platelet-derived growth factor -receptor (PDGFR-) displays a lineage-specific expression pattern in the mouse embryo and is required for normal development of mesoderm and cephalic neural crest derivatives. The purpose of the present study was to demonstrate the in vivo promoter function of genomic DNA fragments representing the 5′-flanking part of the human PDGFRA gene. 2.2, 0.9 and 0.4 kb PDGFRA promoter fragments, ligated to a lacZ reporter gene, were microinjected into fertilized mouse eggs and transgenic mouse lines were established. The expression patterns were basically similar in the 2.2 and 0.9 kb lines and overlapped grossly the endogenous Pdgfra gene expression pattern. The transgenic line with the highest expression level was chosen for detailed analysis. Expression was, as expected, mainly confined to tissues of mesodermal and neural crest origin. No expression was found in epithelial tissues of endo- or ectodermal origin. The promoter fragments were also active in neuroepithelium and in certain neuronal cell types that did not faithfully express PDGFR- mRNA, while they failed to specify reporter expression in PDGFR- expressing O-2A progenitor cells and other glial elements of the central nervous system. Thus, the isolated human PDGFRA promoter contains most but not all of the regulatory elements that are necessary to establish tissue specific gene expression during development.     

Generation and analyses of the transgenic potatoes expressing heterologous thermostable β-amylase

β-Amylase hydrolyzes the -1,4-glycosidic linkages of starch resulting in the release of maltose. This reaction is of industrial importance for maltose production and for the preparation process of fermented foods and alcoholic beverages. A demand for an acceleration of the rate of enzymatic cleavage of the starch macro-molecule is a prerequisite for large-scale and highly efficient production. Increasing the temperature up to the optimum of approximately 60 °C can significantly speed up the reaction. However, at higher temperatures, the effect on protein denaturation becomes dominant, and the conversion rate decreases. The primary objective of this study was to generate transgenic plants of the “Kennebec” potato variety for production of thermostable β-amylase using Agrobacterium-mediated transformation. Four chimeric genes encoding the β-amylase with or without signal peptide sequences for targeting expression in cytoplasm, amyloplasts, or vacuoles were constructed and driven by high tuber expression promoter from Sucrose synthetase gene Sus4. Forty-two transgenic lines were selected for this study. Transgenic lines with various β-amylase constructs were verified for the existence and expression of the transgenes by PCR approaches. The expression level of the introduced β-amylase protein was estimated by immunoblot analyses using polyclonal antibodies. Recombinant β-amylase was successfully expressed in Escherichia coli B21 (DE3), and temperature ranges of these inducible recombinant proteins were found to be between 40 and 90 °C. This enzymatic complex produced in the in vitro cultured microtubers and field-grown tubers from transgenic potatoes were proved to be stable and active at 60 °C. The relative activities of β-amylase in tubers of field-grown potatoes were compared, and the maximum increase was found with transgenic line #6A of the pSUS4-AMY construct which has an 11-fold greater increase than the untransformed “Kennebec”. Variations of the chemical compositions were found in the selected transgenic lines. Results of this study suggest the feasibility of utilizing thermostable β-amylase in transgenic potatoes for the starch-processing industries.     

Regulation of the CYP27B1 5'-flanking region by transforming growth factor-beta in ROS 17/2.8 osteoblast-like cells

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), regulates osteoblast proliferation and differentiation. Production of 1,25(OH)₂D₃ is catalysed by the enzyme 25-hydroxyvitamin D₃-1-hydroxylase (CYP27B1). Though highly expressed in the kidney, the CYP27B1 gene is also expressed in non-renal tissues including bone. It is hypothesised that local production of 1,25(OH)₂D₃ by osteoblasts plays an autocrine or paracrine role. The aim of this study was to investigate what factors regulate expression of the CYP27B1 gene in osteoblast cells. ROS 17/2.8 osteoblast cells were transiently transfected with plasmid constructs containing the 5′-flanking sequence of the human CYP27B1 gene fused to a luciferase reporter gene. Cells were treated with either parathyroid hormone (PTH), 1,25(OH)₂D₃, transforming growth factor-beta (TGF-β) or insulin-like growth factor-1 (IGF-1) and luciferase activity was measured 24 h later. The results showed that 1,25(OH)₂D₃ did not alter expression of the reporter construct, however treatment with PTH, IGF-1 and TGF-β decreased expression by 18, 53 and 58% respectively. The repressive action of TGF-β was isolated to the region between −531 and −305 bp. These data suggest that expression of the 5′-flanking region for the CYP27B1 gene in osteoblast cells may be regulated differently to that previously described in kidney cells.     

Two Pax2/5/8-binding sites in Engrailed2 are required for proper initiation of endogenous mid-hindbrain expression

During early brain development mouse Engrailed2 (En2) is expressed in a broad band across most of the mid-hindbrain region. Evidence from gene expression data, promoter analysis in transgenic mice and mutant phenotype analysis in mice and zebrafish has suggested that Pax2, 5 and 8 play a critical role in regulating En2 mid-hindbrain expression. Previously, we identified two Pax2/5/8-binding sites in a 1.0 kb En2 enhancer fragment that is sufficient to directed reporter gene expression to the early mid-hindbrain region and showed that the two Pax2/5/8-binding sites are essential for the mid-hindbrain expression in transgenic mice. In the present study we have examined the functional requirements of these two Pax2/5/8-binding sites in the context of the endogenous En2 gene for directing mid-hindbrain expression. The two Pax2/5/8-binding sites were deleted from the En2 locus and replaced with the bacterial neo gene by homologous recombination in mouse embryonic stem cells. After transmitting the mutation into mice, the neo gene was removed by breeding with transgenic mice expressing cre from a CMV promoter. Embryos homozygous for this En2 Pax2/5/8-binding site deletion mutation had a mild reduction in En2 expression in the presumptive mid-hindbrain region at the 5-7 somite stage, when En2 expression is normally initiated. However, from embryonic day 9.0 onwards, the mutant embryos showed En2 expression indistinguishable from that seen in wild type embryos. Furthermore, the mutants did not show the cerebellar defect seen in mice with a null mutation in En2. This result demonstrates that the two Pax2/5/8-binding sites that were deleted, while being required for mid-hindbrain expression in the context of a 1.0 kb En2 enhancer, are only required for proper initiation of expression of the endogenous En2 gene. Interestingly, a comparison of the lacZ RNA and protein expression patterns directed by the 1.0 kb enhancer fragment revealed that lacZ protein was acting as a lineage marker in the mid-hindbrain region by persisting longer than the mRNA. The transgene expression directed by the 1.0 kb enhancer fragment therefore does not mimic the entire broad domain of En2 expression. Taken together, these two studies demonstrate that DNA binding sites in addition to the two Pax2/5/8-binding sites must be necessary for En2 mid-hindbrain expression.     

A repressor element in the 5'-untranslated region of human Pax5 exon 1A

Five members of the RecQ helicase family, RECQL, WRN, BLM, RTS and RECQL5, have been found in human and three of them (WRN, BLM and RTS) were disclosed to be the genes responsible for Werner, Bloom and Rothmund–Thomson syndromes, respectively. RECQL5 (RecQ helicase protein-like 5) was isolated as the fifth member of the family in humans through a search of homologous expressed sequence tags. The gene is expressed with at least three alternative splicing products, , β and γ. Here, we isolated mouse RECQL5β and determined the DNA sequence of full-length cDNA as well as the genome organization and chromosome locus. The mouse RECQL5β gene consists of 2949 bp coding 982 amino acid residues. Comparison of amino acid sequence among human (Homo sapiens), mouse (Mus musculus), Drosophila melanogaster and Caenorhabditis elegans RECQL5β homologs revealed three portions of highly conserved regions in addition to the helicase domain. Nineteen exons are dispersed over 40 kbp in the genome and all of the acceptor and donor sites for the splicing of each exon conform to the GT/AG rule. The gene is localized to the mouse chromosome 11E2, which has a syntenic relation to human 17q25.2-q25.3 where human RECQL5β exists. Our genetic characterizations of the mouse RECQL5β gene will contribute to functional studies on the RECQL5β products.     

地衣芽孢杆菌耐高温α-淀粉酶基因的克隆、烟草瞬时表达及转化拟南芥的研究

从地衣芽孢杆菌(Bacillus licheniformis)中克隆到耐高温α-淀粉酶基因全长, 构建了原核表达载体, 转入大肠杆菌(Escherichia coli)中, 使用IPTG于28°C诱导6小时后, 通过SDS-PAGE检测到目的蛋白, 分子量约为55 kDa, 并通过酶活力检测实验证明该蛋白具有耐高温α-淀粉酶活性。同时构建了该基因融合GFP的植物表达载体, 通过农杆菌(Agro- bacterium tumefaciens)介导瞬时转化烟草(Nicotiana tabacum)下表皮细胞并在荧光显微镜下观察, 发现在烟草下表皮细胞的细胞质和液泡中均有绿色荧光。使用I₂-KI溶液对乙醇脱色后的烟草叶片进行染色, 显色反应表明在烟草中表达的耐高温α-淀粉酶具有酶活性。最后, 采用农杆菌介导的花蕾浸泡法将重组载体转化到拟南芥(Arabidopsis thaliana)中, 筛选到稳定遗传的耐高温α-淀粉酶基因的拟南芥纯合子。研究结果为后期开展表达耐高温α-淀粉酶的转基因植物的相关研究奠定了实验基础。