AFM观察小白鼠心肌和肝核DNA片段的基因移位

用原子力显微镜(简称AFM)直接观察、体外表达和体外转录等实验技术组合,观察到了心肌和肝的核DNA片段的基因;用磷酸缓冲液稀释,并用开关蛋白质等活性因子使其部分解离,促使核基因在核DNA片段内或核DNA片段间静态或动态移位,得到了对应核DNA片段中的相关基因,如LDH/DNA体外表达活性变化的LDH同功酶酶谱图。基因静态或动态移位均表达活性降低,且基因移位程度与其对应基因活性降低程度呈正相关性。展示了未来运用AFM和体外表达等实验技术组合研究核DNA片段的基因移位和对应基因突变机制的前景。     

Isolation of a strong matrix attachment region (MAR) and identification of its function in vitro and in vivo

Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR method. The nuclei isolated from suspension- cultured cells of rice were used to prepare nuclear matrix. With a characterized MAR (TM1) as a positive control, the Matrix-MAR interactions were tested by an in vitro binding assay to identify the DNA sequences as MARs and their binding strength to nuclear matrix in vitro was compared. The results showed that TM2 and TM3 had stronger binding strength than TM1. To determine the functions of the four new MARs in vivo, binary vectors pBI121 carrying a uidA GUS reporter gene were modified with direct repeat MARs inserted on both sides of the reporter gene cassette and were transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assays of the transgenic tobaccos showed that when flanking a GUS reporter gene TM1, TM2, TM3 and AM1 increased uidA GUS gene expression level approximately 1.5-fold, 5-fold, 1.35-fold, 1.3-fold respectively and AM2 has no effect on gene expression. TM2 was found to be a strong MAR that could effectively increase gene expression level and could be used as an effective enhancing element to construct high efficient expression vectors. In this note the relations among the sequence features, binding strength in vitro and function in vivo of the five MARs were analyzed, and the potential significance of TM2 in plant genetic engineering was dis- cussed.     

即刻早基因ZENK的基因功能及其应用进展

即刻早基因ZENK是核基因组内编码转录因子的单拷贝基因,它受多种胞外信号,如血清、生长因子、细胞因子和激素的作用在不同类型细胞内迅速、大量表达.ZENK蛋白通过C2H2型锌指识别靶基因启动子中的GC丰富序列并发生作用,从而调控靶基因的转录.ZENK基因参与细胞生长、分化、发育的调控,而且其在大脑的诱导表达与动物的学习、记忆密切关联.另有研究显示,ZENK具有很高的进化保守性,可以作为新的分子标记运用于分子系统学研究.     

Application of a nuclear localization signal gene in transgene mice

Efficient gene transfer by cytoplasm co-injection will offer a powerful means for transgenic animals. Using co-injection in cytoplasm, two independent gene constructs, including bovine α-s1-casein-hG-CSF and a mammal expression vector expressing a nuclear localization signal (mNLS), were introduced into fertilized mouse eggs. The target gene construct was docked into host nucleus probably by the nuclear localization signal. Transgene mice have been obtained at 58% (29/50) of integration ratio. Expression level of the positive transgene mice was detected by Western blotting. Maximal expression of human G-CSF was estimated about 540 mg/L of milk. The expression ratio was up to 75% (9/12). The results here have important practical implications for the generation of mammary gland bioreactors and other transgene studies. Co-injection of a target gene with an expression vector of a mammal nuclear localization signal by cytoplasm appears to be a useful, efficient and easy strategy for generating transgenic animals, which may be able to substitute the routine method of pronucleus-injection of fertilized eggs.     

Isolation of a strong matrix attachment region (MAR) and identification of its function in vitro and in vivo

Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR method. The nuclei isolated from suspensioncultured cells of rice were used to prepare nuclear matrix. With a characterized MAR (TM1) as a positive control, the Matrix-MAR interactions were tested by an in vitro binding assay to identify the DNA sequences as MARs and their binding strength to nuclear matrix in vitro was compared. The results showed that TM2 and TM3 had stronger binding strength than TM1. To determine the functions of the four new MARs in vivo, binary vectors pBI121 carrying a uidA GUS reporter gene were modified with direct repeat MARs inserted on both sides of the reporter gene cassette and were transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assays of the transgenic tobaccos showed that when flanking a GUS reporter gene TM1, TM2, TM3 and AM1 increased uidA GUS gene expression level approximately 1.5-fold, 5-fold, 1.35-fold, 1.3-fold respectively and AM2 has no effect on gene expression. TM2 was found to be a strong MAR that could effectively increase gene expression level and could be used as an effective enhancing element to construct high efficient expression vectors. In this note the relations among the sequence features, binding strength in vitro and function in vivo of the five MARs were analyzed, and the potential significance of TM2 in plant genetic engineering was discussed.     

Chloroplast to nucleus communication triggered by accumulation of Mg-protoporphyrinIX

Plant cells coordinately regulate the expression of nuclear and plastid genes that encode components of the photosynthetic apparatus. Nuclear genes that regulate chloroplast development and chloroplast gene expression provide part of this coordinate control. There is evidence that information also flows in the opposite direction, from chloroplasts to the nucleus. Until now, at least three different signalling pathways have been identified that originate in the plastid and control nuclear gene expression but the molecular nature of these signals has remained unknown. Here we show that the tetrapyrrole intermediate Mg-protoporphyrin (Mg-ProtoIX) acts as a signalling molecule in one of the signalling pathways between the chloroplast and nucleus. Accumulation of Mg-ProtoIX is both necessary and sufficient to regulate the expression of many nuclear genes encoding chloroplastic proteins associated with photosynthesis.     

Homology of a candidate spermatogenic gene from the mouse Y chromosome to the ubiquitin-activating enzyme E1.

The Sxr (sex-reversed) region, a fragment of the Y chromosome short arm, can cause chromosomally female XXSxr or XSxrO mice to develop as sterile males. The original Sxr region, termed Sxra, encodes: Tdy, the primary sex-determining gene; Hya, the controlling or structural locus for the minor transplantation antigen H-Y; gene(s) controlling the expression of the serologically detected male antigen (SDMA); Spy, a gene(s) required for the survival and proliferation of A spermatogonia during spermatogenesis; Zfy-1/Zfy-2, zinc-finger-containing genes of unknown function; and Sry, which is probably identical to Tdy. A deletion variant of Sxra, termed Sxrb, which lacks Hya, SDMA expression, Spy and some Zfy-2 sequences, makes positional cloning of these genes possible. We report here the isolation of a new testis-specific gene, Sby, mapping to the DNA deleted from the Sxrb region (the delta Sxrb interval). Sby has extensive homology to the X-linked human ubiquitin-activating enzyme E1. The critical role of this enzyme in nuclear DNA replication together with the testis-specific expression of Sby suggests Sby as a candidate for the spermatogenic gene Spy.     

9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha.

Vitamin A (retinol) and its natural derivatives are required for many physiological processes. The activity of retinoids is thought to be mediated by interactions with two subfamilies of nuclear retinoic acid receptors, RAR and RXR. The RARs bind all-trans retinoic acid (t-RA) with high affinity and alter gene expression as a consequence of this direct ligand interaction. RXR alpha is activated by t-RA, yet has little binding affinity for this ligand. t-RA may be converted to a more proximate ligand that directly binds and activates RXR alpha, and we have developed a method of nuclear receptor-dependent ligand trapping to test this hypothesis. Here we report the identification of a stereoisomer of retinoic acid, 9-cis retinoic acid, which directly binds and activates RXR alpha. These results suggest a new role for isomerization in the physiology of natural retinoids.     

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic loach (Misgurnus anguillicaudatus Cantor) have been studied. TheGFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of theGFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.     

甜菜夜蛾多核壳型核型多角体病毒vp39基因的克隆与表达

参照苜宿银纹夜蛾核型多角体病毒（AcMNPV)衣壳蛋白基因vp39的序列设计引物,采用PCR技术扩增了甜菜夜蛾核型多角体病毒的约1．3kb片段,通过将片段亚克隆至原核表达载体pET-28构建出重组表达质粒pET-Se39,以pET-Se39转化E.coli BL21。经IPTG诱导后,SeMNPVvp39基因高效表达,SDS－PAGE分析显示表达产物的分子量为39KD,并且表达量在IPTG诱导4h达到最高水平。／