Sequences in the proximal 5′ flanking region of the rat neuron-specific enolase (NSE) gene are sufficient for cell type-specific reporter gene expression

We investigated the regulation of the rat neuron-specific enolase gene using a transient transfection approach. Recent transgenic mouse studies have shown that a 1.8-kb segment of the ratNSE gene 5′ flanking region, including the first (noncoding) exon but not the first intron, is able to drive expression of a reporter gene in parallel with endogenousNSE. These data suggest thatcis-acting elements responsible for the spatial and temporal pattern ofNSE gene expression are located within the proximal 1.8 kb of the 5′ flanking sequence. To further investigate this region, we joined the 1.8-kb regulatory cassette to thecat reporter gene and generated a number of constructs in which the flanking sequence was progressively deleted from the 5′ end. These constructs were tested by transient transfection into neuronal and nonneuronal cells, followed by an assay for CAT activity. We found that as little as 255 bp of 5′ flanking sequence was able to confer cell type-specificity on the reporter gene. Further truncation to 120 bp of 5′ sequence resulted in a sharp downregulation of reporter activity in PC12 cells but a significant rise in both Neuro-2A neuroblastoma cells and nonneuronal Ltk- cells, indicating thatcis-acting elements controlling the regulation ofNSE in Ltk-, Neuro-2A, and PC12 cells may lie within the 135 bp region covered by this deletion. This region contains an AP-2 site and an element similar in sequence and position to a motif identified in the proximal promoter region of the neuron-specific peripherin gene. Reduction to 95 bp of 5′ sequence resulted in a slight downregulation of CAT activity in all cell lines tested, and further truncation to 65 bp of 5′ sequence caused a universal reduction to background levels of CAT activity, concomitant with the disruption of the basalNSE promoter. Our results show that the 5′ flanking region of theNSE gene is capable of conferring cell type-specificity on a heterologous gene in transfected cells and that elements responsible for this are located within the proximal 255 bp.     

视蛋白基因启动子的克隆和鉴定

目的 克隆出视蛋白基因启动子。方法 以小鼠全基因组为模板，用PCR法克隆出目的大小的片断。然后连接到T-载体上作酶切鉴定，最后测序。结果 酶切结果与预期相符，测序结果与公布序列完全一致。结论 视蛋白基因启动子克隆成功。     

Kringle 5的真核植物细胞穿梭表达载体的构建

目的：构建含有目的基因kring5的真核植物细胞穿酸表达载体。方法：应用RT－PCR方法，从正常人肝脏组织中获得kringle5基因，测序后，再通过DNA重组技术，将kringle5基因片段克隆到真核植物细胞表达载体pBI121和pCAMBIA3301上。结果：得到的kringle5基因序列测定结果与文献相符，重组载体pB1k5和pC33k5经酶切鉴定正确，含有植物高效表达CaMV35S启动子。结论：重组载体pB1k5和pC33k5不仅可以在大肠杆菌中稳定复制，而且可以在植物细胞中表达。     

小鼠足容积的活体和离体测量

本实验建立小鼠足容积测量方法学并提供小鼠足容积的生理数值，为进一步研究实验性皮肤癌发生的免疫机制打下基础．根据毛细管放大原理，使用自制的小鼠足容积测量仪测量３５１只昆明种小鼠左右足容积，发现左右足容积几乎相等．另对１５０只小鼠左右足进行３６９次重复测量，发现合格测量超过９７％．不同体重小鼠足生长曲线研究表明，随着体重的增加，小鼠足容积渐趋于一定值，表明生长停止．并且体重２５ｇ以上小鼠活体离体足容积差接近一定值，这提示大龄小鼠足血管张力和容积基本恒定．     

A cytoskeletal actin gene in the mosquito Anopheles gambiae

Five actin genes have been identified in the mosquito Anopheles gambiae , and a constitutively expressed actin gene has been chosen for detailed analysis. We have physically mapped and sequenced this gene and six associated cDNAs, including translated coding regions, as well as the 5 and 3 flanking sequences. Analysis of stage-specific RNA shows this gene to be present in all stages of mosquito development and in an established A. gambiae cell line, thus indicating a cytoskeietal actin. In the sequence of the translated coding region and in pattern of expression, this gene is very similar to the cytoskeietal actin genes of Droso-phila melanogaster , and in sequence, equally similar to the Artemia cytoskeietal actin gene 403 (99.2% identity among the three amino acid sequences). Sequencing of this A. gambiae actin gene (designated actWior its location in chromosome division 1D) and selected cDNAs shows that it possesses three alternative leader sequences; thus the gene appears to have three alternative promoters. These promoters should ultimately prove useful in the production of transgenic constructs for constitutive expression.     

FasL启动子调控的报告基因表达质粒的构建及活性分析

目的扩增FasL启动子并构建带有FasL启动子的荧光素酶报告质粒,并评价在皮质酮(啮齿类动物体内的糖皮质激素)作用的睾丸Leydig细胞中,由FasL启动子调控的荧光素酶的表达活性。本研究将为进一步分析Leydig细胞中FasL启动子转录调控的作用机制奠定基础。方法采用DNA重组技术构建由FasL启动子调控的荧光素酶报告基因的表达质粒。此重组质粒经阳离子脂质体LipofectAMINE介导,瞬时转染入Leydig细胞中,36h后细胞经皮质酮处理,并于48h时收集细胞。测定荧光素酶的相对表达活性。结果(1)酶切及测序证实成功扩增FasL基因启动子并构建了带有FasL基因启动子的报告基因表达质粒；(2)皮质酮处理的Leydig细胞中FasL启动子调控的荧光素酶表达质粒的表达活性显著增高。结论构建成功的带有FasL启动子的荧光素酶报告质粒可准确地评价FasL启动子在凋亡过程中的作用。