Negative regulatory element associated with potentially functional promoter and enhancer elements in the long terminal repeats of endogenous murine leukemia virus-related proviral sequences.

Three series of recombinant DNA clones were constructed, with the bacterial chloramphenicol acetyltransferase (CAT) gene as a quantitative indicator, to examine the activities of promoter and enhancer sequence elements in the 5' long terminal repeat (LTR) of murine leukemia virus (MuLV)-related proviral sequences isolated from the mouse genome. Transient CAT expression was determined in mouse NIH 3T3, human HT1080, and mink CCL64 cultured cells transfected with the LTR-CAT constructs. The 700-base-pair (bp) LTRs of three polytropic MuLV-related proviral clones and the 750-bp LTRs of four modified polytropic proviral clones, in complete structures either with or without the adjacent downstream sequences, all showed very little or negligible activities for CAT expression, while ecotropic MuLV LTRs were highly active. The MuLV-related LTRs were divided into three portions and examined separately. The 3' portion of the MuLV-related LTRs that contains the CCAAC and TATAA boxes was found to be a functional promoter, being about one-half to one-third as active as the corresponding portion of ecotropic MuLV LTRs. A MboI-Bg/II fragment, representing the distinct 190- to 200-bp inserted segment in the middle, was found to be a potential enhancer, especially when examined in combination with the simian virus 40 promoter in CCL64 cells. A PstI-MboI fragment of the 5' portion, which contains the protein-binding motifs of the enhancer segment as well as the upstream LTR sequences, showed moderate enhancer activities in CCL6 cells but was virtually inactive in NIH 3T3 cells and HT1080 cells; addition of this fragment to the ecotropic LTR-CAT constructs depressed CAT expression. Further analyses using chimeric LTR constructs located the presence of a strong negative regulatory element within the region containing the 5' portion of the enhancer and the immediate upstream sequences in the MuLV-related LTRs.     

Tissue restricted and stage specific transcription is maintained within 411 nucleotides flanking the 5'' end of the chicken alpha-skeletal actin gene.

alpha-skeletal actin message levels have been shown to be tightly regulated in chicken primary myoblast cultures. To test for gene elements required for muscle cell specific expression, DNA sequences containing the 5'-flanking regions of the chicken alpha-skeletal actin, beta-cytoplasmic actin, and the histone H2b genes were linked to the coding sequences of the chloramphenicol acetyltransferase gene and transfected into myogenic and non-myogenic cells. In contrast to beta-actin CAT hybrids, the alpha-skeletal actin CAT constructions displayed restricted CAT expression in transfected non-myogenic cells. We showed that a 411 nucleotide fragment flanking the 5' end of of the alpha-skeletal actin gene was responsible for a 9-15 fold increase in CAT enzymatic activity during myoblast fusion, versus only a transient 2 fold rise for the beta-actin and histone flanking sequences. These results indicate that DNA sequences within 411 bp of the 5' terminus of the alpha-skeletal actin gene influenced its cell type and stage specific expression.     

Characterization of the 5' flanking region of rat glucokinase gene

The rat glucokinase (GK) gene containing the first exon was isolated and its 5' flanking region was characterized by the bacterial chloramphenicol acetyltransferase (CAT) assay. A transient expression assay with a series of 5' deletion constructs (-5.5 k to -48) of GK-CAT fusion genes indicated that the 5' flanking sequence up to nucleotide -87 was sufficient for promoter activity in adult rat hepatocytes, but its activity was much weaker than that of the SV40 enhancer/promoter. Similar promoter activity was also detected in dRLh-84 hepatoma cells, which do not express glucokinase. Insulin treatment caused no change in the CAT activity of hepatocytes transfected with the fusion genes. These results suggest that the 5' flanking region of the glucokinase gene up to -5.5 k does not contain enhancer elements responsible for tissue-specific expression or insulin regulation.     

人淋巴细胞CD2基因5’侧翼序列的克隆和鉴定

本文报告以ＣＤ２ｃＤＮＡ５’端的片段作探针,从人Ｔ淋巴细胞基因组文库筛选阳性重组克隆,经限制性内切酶降解和Ｓｏｕｔｈｅｒｎ杂交分析,证明其中一个阳性克隆的插入片段中含ＣＤ２基因５’侧翼顺序。经插入片段的亚克隆、限制性内切酶图谱及ＤＮＡ序列分析,鉴定出一含转录起始点及其上游序列的４．０ｋｂ片段。将此片段中含转录起始点和两个ＤＮ（ａｓｅ）Ⅰ高敏感位点的２．５ｋｂ片段定向克隆到以虫萤光素酶为报告基因的表达载体ｐＭＧ３中,并用限制性内切酶对此２．５ｋｂ片段作不同程度缺失,构成一系列突变子。这些重组的表达质粒转染人ＪｕｒｋａｔＴ细胞后,以瞬时表达实验分析各突变子驱动虫萤光素酶基因的表达,结果发现在ＣＤ２基因５’上游具有很弱的启动子活性,初步测定该启动子位于－１．２ｋｂ～－９８ｂｐ域。ＣＤ２基因具有弱启动子、强增强子的特点与Ｔ细胞表面其它抗原分子基因是相似的。     

Expression of the K-fgf proto-oncogene is controlled by 3'' regulatory elements which are specific for embryonal carcinoma cells.

Expression of the K-fgf/hst proto-oncogene appears to be restricted to cells in the early stages of development, such as embryonal carcinoma (EC) cells. When EC cells are induced to differentiate, K-fgf expression is drastically repressed. To identify cis-acting DNA elements responsible for this type of regulation, we constructed a plasmid in which cat gene expression was driven by about 1 kilobase of upstream K-fgf human DNA sequences, including the putative promoter, and transfected it into undifferentiated F9 EC cells or HeLa cells as prototypes of cells which express or do not express, respectively, the K-fgf proto-oncogene. This plasmid was essentially inactive in both cell types, and the addition of more than 8 kilobases of DNA sequences upstream of the K-fgf promoter did not lead to any increase in chloramphenicol acetyltransferase (CAT) expression. On the other hand, when we inserted in this plasmid DNA sequences which are 3' of the human K-fgf coding sequences, we could detect a significant stimulation of CAT activity. Analysis of these sequences led to the identification of enhancerlike DNA elements which are part of the 3' noncoding region of K-fgf exon 3 and promote CAT expression only in undifferentiated mouse F9 or human NT2/D1 EC cells, but not in HeLa, 3T3, or differentiated F9 cells, therefore mimicking the physiological expression of the K-fgf proto-oncogene. Similar elements are also present in the 3' region of the murine K-fgf proto-oncogene, in a region showing high homology to the human K-fgf sequences. These regulatory elements can promote CAT expression from heterologous promoters in an EC-specific manner, suggesting that they interact with a specific cellular transacting protein(s) whose expression is developmentally regulated.