Tissue specificity of enhancer and promoter activities of a HERV-K(HML-2) LTR

Transient expression of a luciferase reporter gene was used to evaluate tissue-specific promoter and enhancer activities of a solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K) in several human and CHO cell lines. The promoter activity of the LTR varied from virtually not detectable (GS and Jurkat cells) to as high as that of the SV40 early promoter (Tera-1 human testicular embryonal carcinoma cells). The negative regulatory element (NRE) of the LTR retained its activity in all cell lines where the LTR could act as a promoter, and was also capable of binding host cell nuclear proteins. The enhancer activity of the LTR towards the SV40 early promoter was detected only in Tera-1 cells and was not observed in a closely related human testicular embryonal carcinoma cell line of different origin, NT2/D1. A comparison of proteins bound to central part of the LTR in nuclear extracts from Tera-1 and NT2/D1 by electrophoretic mobility shift assay revealed striking differences that could be determined by different LTR enhancer activities in these cells. Tissue specificity of the SV40 early promoter activity was also revealed.     

Genomic sequence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) KLV-2010-01 originating from the first Korean outbreak in cultured Litopenaeus vannamei

Due to the need to track and monitor genetic diversity, the genome of the infectious hypodermal and hematopoietic necrosis virus (IHHNV) strain KLV-2010-01 in cultured Litopenaeus vannamei shrimp that originated from the first Korean outbreak in 2010 was sequenced and analyzed. The genome, with a length of 3914 nucleotides, was sequenced from the Korean IHHNV. The genome encoded three large and overlapping open reading frames: ORF1 (NS-1) of 2001 bp, ORF2 (NS-2) of 1092 bp and ORF3 (capsid protein) of 990 bp. The overall organization, size and predicted amino acid sequence of the three ORFs in Korean IHHNV were highly similar to those of members of the infectious IHHNV group, and the most closely related strains were IHHNVs described from Ecuador and Hawaii. Additionally, phylogenetic analysis showed that the Korean IHHNV was clustered with lineage III in the infectious IHHNV group and was most similar to IHHNV isolates from Ecuador, China and Taiwan.     

Transcriptional mapping and nucleotide sequence of the Listeria monocytogenes hlyA region reveal structural features that may be involved in regulation

DNA sequence analysis of the regions adjacent to the hlyA gene, which encodes listeriolysin O, an essential virulence factor of Listeria monocytogenes, revealed the presence of two open reading frames (ORFs): ORF D located 304 base pairs downstream from hlyA, and ORF U located 224 base pairs upstream from and in opposite direction to hlyA. Promoter mapping performed with RNAs extracted from cells growing exponentially in rich medium showed that the three ORFs are independently transcribed. hlyA is transcribed from two promoters separated by 10 base pairs (P1 hlyA and P2 hlyA). ORF U is transcribed in the opposite direction from an adjacent promoter. These two promoter regions are separated by a palindromic sequence T-T-A-A-C-A-A/T-T-G-T-T-A-A. This palindrome was also found upstream from the ORF D promoter, suggesting that all three genes are similarly regulated.     

鼻咽癌细胞中ezrin基因启动子上游序列转录调控特性的研究

目的:研究鼻咽癌细胞中ezrin基因启动子上游序列的转录调控特性。方法:构建一系列携带ezrin基因-1541/-706序列的报告基因表达载体,ezrin基因-1541/-706序列以正向和反向分别连接至不含启动子的报告基因上游、ezrin启动子上游、SV40启动子上游、ezrin启动子或SV40启动子控制的报告基因下游,将质粒转染CNE2细胞,检测荧光素酶活性。结果:CNE2细胞中,当-1541/-706序列正向位于报告基因上游时表现出启动子活性,其转录激活作用约为ezrin启动子的50%;反向连接时无启动子活性。而且,当-1541/-706序列正向位于ezrin启动子或SV40启动子上游时,显著提高荧光素酶表达;当反向位于启动子下游、正向或反向位于启动子控制的报告基因下游时,转录增强作用消失。结论:CNE2细胞中ezrin基因启动子上游序列具有转录激活和转录增强作用,这种作用具有DNA序列位置和方向依赖性。     

Analysis of the early regulatory region of the human papovavirus BK

BKV is a human papovavirus which latently infects a majority of the world population and whose DNA has been found in human tumor tissue. Along with simian virus 40 (SV40) and JCV, it is one of several highly homologous polyomaviruses which display distinct host ranges, tissue tropisms, and transformation potentials. Determination of these properties is thought to reside, in part, in the noncoding regulatory region of these viruses. We have studied the regulation of gene expression by the early promoter and enhancer of BKV. Our results show that the early promoter of BKV consists of elements found both to the early side of and within the proximal 18 bp of the first enhancer element itself. At least one BKV regulatory element appears to be downstream of the mRNA start sites. The BKV enhancer consists of two different types of elements, three direct repeats, and an element (denoted "c") found in the 30 bp to the late side of the distal repeat. When used with the BKV promoter the enhancer repeat elements were found to be redundant, optimal promoter activity requiring only two of the three repeats plus the c element. Using the heterologous SV40 promoter the optimal BKV enhancer consisted of either three repeats or two repeats plus the c element. Subfragments of the enhancer region were capable of partial activation of homologous and heterologous promoters. We conclude that the regulation of BKV early gene expression involves novel elements arranged in ways not previously described in other papovaviruses.     

CKLF基因与CKLFSF1基因间的顺式调控元件

目的：探讨CKLF基因与CKLFSF1基因间序列(CCS)的调控作用。方法：运用PCR技术扩增CCS，并将此片段插入含有荧光素酶(luriferase)报告基因载体pGL3-basic，以及pGL3-SV40启动子中，构建受其调控的荧光素酶报告基因载体。应用脂质体介导基因转染技术，将4种重组质粒转染Hela细胞，进行瞬时表达分析。结果：在pGL3-basic和pGL3-basic-CCS质粒中的报告基因luciferase均无表达，但将CCS片段插入至promoter上游后，荧光素酶活性增加近1倍。结论：CKLF与CKLFSF1基因间的序列不具有启动子活性，但该序列中却可能存在调控其下游基因表达的顺式增强子元件。     

纤维连结蛋白启动子的克隆及转录活性的研究

目的：克隆纤维连结蛋白（FN） 启动子和检测其转录活性，并初步研究增加SV40增强子对其转录活性的影响。方法:从正常人gDNA中克隆FN启动子，驱动氯霉素乙酰转移酶报告基因表达，构建成有SV40增强子和无SV40增强子两个重组体报告载体。利用脂质体介导的转染技术导入HT1080细胞，检测各重组体的瞬时表达，确定克隆FN启动子转录活性和SV40增强子对其转录活性的影响。 结果:克隆的FN启动子在HT1080细胞中活性比SV40启动子稍强，在加入SV40增强子时活性提高了约6倍。 结论:成功的克隆FN启动子，在HT1080细胞中有活性，通过增加SV40增强子能大大提高其活性，为运用FN启动子和特异性靶细胞治疗各种遗传性疾病提供了一定基础。     

Baculovirus Expression of Proteins of Porcine Reproductive and Respiratory Syndrome Virus Strain Olot/91. Involvement of ORF3 and ORF5 Proteins in Protection

Porcine reproductive and respiratory syndrome virus (PRRSV) is a new arterivirus that has spread rapidly all around the world in the last few years. The genomic region containing open reading frames (ORFs) 2 to 7 of PRRSV Spanish isolate Olot/91 was cloned and sequenced. The genomic sequence shared 95% identity with Lelystad and Tu¨bingen isolates and between 61–64% with the ORF7 region of the American isolates. ORFs 2 to 7 were inserted into recombinant baculoviruses downstream of the polyhedrin promoter. Only ORFs 2, 3 5 and 7 were expressed in insect cells as detected by PRRS-specific pig antisera. To analyze the immunogenicity of these proteins and their ability to confer protection, Sf9 cells infected with recombinant baculoviruses expressing ORFs 3, 5 and 7 gene products were used to immunize pregnant sows, either individually or in combination. The results obtained indicate that ORFs 3 and 5 gene products could be major candidates for the development of a vaccine against PRRS since they conferred 68.4 and 50% protection, respectively, as evaluated by the number of piglets born alive and healthy at the time of weaning. In addition, piglets born to sows immunized with ORFs 3 and 5 proteins were seronegative to PRRSV after weaning, indicating absence of viral replication. ORF7 is the most immunogenic protein of PRRSV, but the antibodies induced in sows are non-protective and may even interfere with protection.     

活化T细胞核因子对组成性启动子SV40的调控作用

目的:利用双荧光素酶报告系统探讨活化T细胞核因子(NFAT)能否调控常见组成性启动子CMV,SV40和TK,为不同条件下选择合适双荧光报告系统内参提供依据。方法:用限制性内切酶BglⅡ和HindⅢ分别从质粒pCDNA3.1和pRL-TK中切下CMV和TK启动子,克隆至pGL3-basic载体中,构建成pCMV-Luc和pTK-Luc载体。将构建pCMV-Luc和pTK-Luc以及商品化的pGL3-control(SV40启动子驱动),分别与SV40(pBIND)和TK(pRL-TK)两种启动子驱动的两种内参质粒共转染入HEK293细胞;观察过表达组成性活化NFAT后相对荧光素酶活性读数的改变。结果:成功构建了pCMV-Luc和pTK-Luc质粒,荧光素酶活性检测发现,常见组成性启动子SV40启动子对过表达组成性活化的NFAT存在一定的反应。结论:T细胞活化过程中重要的转录因子NFAT能够调控SV40启动子活性;表明常见组成性启动子SV40并非真正、绝对的组成性不变。因此,在荧光素酶报告系统内参选择时需要充分考虑该问题,本研究为合理选择内参质粒提供了一个可行策略。