Depression of the xylanase-encoding cgxA gene of Chaetomium gracile in Aspergillus nidulans

Regulation of the Chaetomium gracile xylanase A gene (cgxA) was investigated using Aspergillus nidulans as an intermediate host. Deletion of a 185 bp DNA fragment from its promoter region led to higher levels of the cgxA gene expression, indicating that the 185 bp DNA fragment contains an element involved in repression of the gene. A nuclear extract was assayed for proteins which bind to the 185 bp DNA fragment. A protein designated AnRP bound sequence specifically to the DNA fragment. The minimum sequence required for AnRP binding, 5'TTGACAAAT-3', was determined by means of gel mobility shift assays with various double-stranded oligonucleotides. Furthermore, this sequence repressed the expression of the cgxA gene when inserted at the 5' end of the cgxA gene on pXAH, which was deleted for the repressive element from the promoter region.     

稻瘟病菌(Magnaporthe grisea)诱导的水稻早期反应基因ER1的5' 端cDNA片段克隆及序列分析

研究高等生物基因表达与调控的一个重要方面是分离基因的编码区及其上游的调控序列（ＤｅＶｅｅｒ等１９９７）,这需要获得一个基因的ｃＤＮＡ全长及从植物基因组获取全基因。在前文（周建明等１９９９）中曾经分离了稻瘟病菌侵染诱导的水稻早期反应基因ＥＲ１的ｃＤＮＡ片段,但是运用ｍＲＮＡ差异显示技术分离的ｃＤＮＡ片段往往只有近ｍＲＮＡ３’端的一部分,难以反映基因的结构及功能特点,因此,必须进一步分离其５’端的部分才有可能比较全面地了解此基因的特点。ＲＡＣＥ（ｒａｐｉｄａｍｐｌｉｆｉｃａｔｉｏｎｏｆｃＤＮＡｅｎ…     

Characterization of the 5' flanking region of the rat Na+/K(+)-ATPase beta 1 subunit gene.

We have isolated the 5' end of the rat Na+/K(+)-ATPase beta 1 subunit gene. A genomic fragment containing 817 bp of the 5' flanking sequence, exon 1 and 479 bp of intron 1 was sequenced. The 5' flanking region contains a potential TATA box and several putative CAAT and GC boxes. Potential binding sites for thyroid and glucocorticoid receptors were identified together with multiple sequence motifs which exhibit homology to calcium and serum responsive elements.     

Promoter analysis of tumor suppressor gene PTEN: identification of minimum promoter region

Mutations of PTEN, a tumor suppressor gene located on chromosome 10, which encodes a protein-tyrosine and lipid-phosphatase, are prevalent in various human cancers, including glioblastoma. Despite extensive characterization of PTEN mutations in human cancers and a relatively good understanding of the molecular roles of PTEN in the control of cellular processes, little is known about modes of PTEN regulation. To understand the regulation of expression of the tumor suppressor gene PTEN, we isolated a 2212 bp fragment from the human BAC clone 46B12 DNA. The 3' end of this fragment starts at the Not I site of -745 relative to the first translation codon ATG (+1) and ends at the Sal I site of -2957 at the 5' end. Using classical 5'RACE and primer extension techniques, nine start sites were observed between -817 and -984 upstream of the ATG start site. We located a 137 bp fragment (-958/-821) as the minimum promoter region using promoter deletion and luciferase assays. A 704 bp fragment (-33/-737) downstream of the 2212 bp fragment was also cloned. As indicated by luciferase assays, the data show that this region possesses no promoter function. Interestingly, a p53 binding sequence is located within the 599 bp fragment (-1344/-745), although p53 expression had a minimal effect on PTEN, demonstrating its insignificant role in PTEN gene expression.     

The human urokinase-plasminogen activator gene and its promoter.

The urokinase type of plasminogen activator (uPA) is subject to regulation by hormones, phorbol esters and oncogenic transformation. This enzyme has been suggested to play a key role in processes involving cell migration and tissue remodeling, and to be essential for tumor metastasis. In order to study these processes, we have isolated the human uPA gene, and have determined its entire nucleotide sequence. The gene is organized in 11 exons and is 6.4 kb long. The 5' end of uPA mRNA has been determined by both S1 mapping and primer extension experiments. A fragment of 800 bp containing the entire 5' flanking region shows promoter activity when introduced upstream of a bacterial chloramphenicol acetyltransferase gene and introduced into human cells. The hexanucleotide sequence GGCGGG, previously found at similar regions in several viral and eukaryotic promoters and shown to be essential for promoter activity (McKnight et al. (1984) Cell, 37, 253-262), is repeated three times between the CAAT and the TATA boxes.     

Sequence of a sea urchin hsp70 gene and its 5' flanking region

We report the nucleotide sequence of a 4470-bp fragment derived from a sea urchin genomic clone containing part of a heat-shock protein 70 (Hsp70)-encoding gene. This fragment, named hsp70 gene II, contains 1271 bp of the flanking region and 3299 bp of structural gene sequence interrupted by five introns and encoding the N-terminal 371 amino acids (aa) of the protein. The 5' flanking region contains a putative TATA element, two CCAAT boxes, four heat-shock consensus sequence elements (hse) and one consensus sequence for binding of Sp1. Remarkable homologies were observed for deduced aa sequence and intron-exon organization between hsp70 gene II and rat hsc73 gene.     

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。