RIP60, a mammalian origin-binding protein, enhances DNA bending near the dihydrofolate reductase origin of replication.

Replication of the Chinese hamster dihydrofolate (dhfr) gene initiates near a 281-bp HaeIII fragment of stably bent DNA that binds RIP60, a 60-kDa origin-specific DNA-binding protein that has been purified from HeLa cell nuclear extract (L. Dailey, M. S. Caddle, N. Heintz, and N. H. Heintz, Mol. Cell. Biol. 10:6225-6235, 1990). Circular permutation assays showed that stable DNA bending in the dhfr origin region fragment was due to the presence of five oligo (dA)3-4 tracts, designated bend elements B1 to B5, that are spaced 10 bp apart. DNA bending directed by elements B1 to B5, as assessed by anomolous migration of DNA fragments on polyacrylamide gels, was accentuated at 4 degrees C. Bend element B5, which is in inverse orientation relative to elements B1 to B4, overlaps an ATT-rich motif that comprises the RIP60 protein-binding site. Gel mobility shift assays with circularly permuted bent DNA fragments and purified RIP60 showed that RIP60 markedly enhanced DNA bending of the dhfr origin region sequences. These results suggest that, as in many plasmids, bacteriophages, and eucaryotic viruses, mammalian DNA-binding proteins may enhance DNA bending near origins of replication during initiation of DNA synthesis.     

Replication origins oriGNAI3 and oriB of the mammalian AMPD2 locus nested in a region of straight DNA flanked by intrinsically bent DNA sites

The aim of this work was to determine whether intrinsically bent DNA sites are present at, or close to, the mammalian replication origins oriGNAI3 and oriB in the Chinese hamster AMPD2 locus. Using an electrophoretic mobility shift assay and in silico analysis, we located four intrinsically bent DNA sites (b1 to b4) in a fragment that contains the oriGNAI3 and one site (b5) proximal to oriB. The helical parameters show that each bent DNA site is curved in a left-handed superhelical writhe. A 2D projection of 3D fragment trajectories revealed that oriGNAI3 is located in a relatively straight segment flanked by bent sites b1 and b2, which map in previously identified Scaffold/Matrix Attachment Region. Sites b3 and b4 are located approximately 2 kb downstream and force the fragment into a strong closed loop structure. The b5 site is also located in an S/MAR that is found just downstream of oriB.     

Detection, sequence patterns and function of unusual DNA structures.

Unusual DNA structures were detected by an electrophoretic procedure in which DNA fragments were separated according to size on agarose gels and then by shape on polyacrylamide gels. Fragments from yeast centromeres migrated faster in polyacrylamide than predicted from their base composition and size and this property was attributed to a nonrandom distribution of oligomeric A tracts that exhibited minima at 10-11 base intervals. Fragments from seven loci in 107 kb of DNA migrated anomalously slow and these fragments contained blocks of A2-6 in a 10-11 base periodicity which is indicative of bent DNA. The most pronounced bent sequences were found within yeast ARS1 and centered at 245 and 240 bp from the left and right ends of the adenovirus genome. Each sequence is approximately 150 bp away from a replication origin and the adenovirus sequences are within 50 bp of enhancers. Nuclear matrix attachment sites, which are also adjacent to enhancers, contain sequences characteristic of bent DNA. These results suggest that bent structures reside at the base of DNA loops in chromosomes.     

Intrinsically bent DNA in the promoter regions of the yeast GAAL1-10 and GAL80 genes

Circular permutation analysis has detected fairly strong sites of intrinsic DNA bending on the promoter regions of the yeast GAL1-10 and GAL80 genes. These bends lie in functionally suggestive locations. On the promoter of the GAL1-10 structural genes, strong bends bracket nucleosome B, which lies between the UAS(G) and the GAL1 TATA. These intrinsic bends could help position nucleosome B. Nucleosome B plus two other promoter nucleosomes protect the TATA and start site elements in the inactive state of expression but are completely disrupted (removed) when GAL1-10 expression is induced. The strongest intrinsic bend ( approximately 70 degrees ) lies at the downstream edge of nucleosome B; this places it approximately 30 base pairs upstream of the GAL1 TATA, a position that could allow it to be involved in GAL1 activation in several ways, including the recruitment of a yeast HMG protein that is required for the normally robust level of GAL1 expression in the induced state (Paull, T., Carey, M., and Johnson, R. (1996) Genes Dev. 10, 2769-2781). On the regulatory gene GAL80, the single bend lies in the non-nucleosomal hypersensitive region, between a GAL80-specific far upstream promoter element and the more gene-proximal promoter elements. GAL80 promoter region nucleosomes contain no intrinsically bent DNA.     

雌激素通过LRP16基因5′侧翼GC富含区上调其转录的分子机制

LRP16是一个明确的雌激素(E2)反应性靶基因,已往研究在LRP16基因上游调控区鉴定了一个E2反应性1/2ERE/GC富含的ERα作用位点（-676 bp到-214 bp;命名为A区）.为进一步鉴定雌激素上调LRP16基因表达的最大化反应区域,对LRP16基因上游调控区进行缺失突变,通过相对荧光素酶活性分析观察到LRP16基因的一段5′近端侧翼序列（-213 bp到-24 bp;命名为B区）具有明显的E2反应性.通过与A区比较,认为B区最大化的呈递了E2对LRP16基因的转激活效应.序列分析表明,B区缺乏经典的ERE元件,而包含多个富含GC序列.针对Sp1的siRNA实验结果提示,Sp1参与了E2对该区域的转激活.针对GC富含区进一步的缺失突变,及荧光素酶活性分析,识别了一段30 bp（-213 bp到-184 bp）的序列在B区呈递E2反应活性中发挥核心作用.超级凝胶电泳实验结果表明,Sp1蛋白与这段30 bp的DNA序列在体外存在直接结合作用.染色质免疫共沉淀实验结果证实,ERα、Sp1与B区存在E2依赖性相互作用.本文在LRP16的5′侧翼区识别了一段最大化呈递E2活性的DNA片段.机制研究表明,在E2存在条件下,ERα通过Sp1与该区域的直接作用上调LRP16基因的表达.     

Unusual DNA structures in the adenovirus genome

More than 80% (approximately 29 kilobase pairs) of the adenovirus serotype 2 genome was surveyed for the presence of unusual DNA conformations. Seven recombinant DNAs containing the largest HindIII fragments of AD2 DNA were analyzed for the presence of negative supercoil-dependent S1 nuclease-sensitive sites. Four plasmids each contained a specific site of S1 nuclease sensitivity whereas the other three showed no reaction. Further investigation was focused on a plasmid containing one of the positively reacting fragments (fragment C) which contained the major late promoter at coordinate 16.4 on the genome; three serotypes (Ad2, Ad7, Ad12) were studied. Fine mapping studies revealed the S1-sensitive sites to be a small region (approximately 6 base pairs) located at the TATA box of the major late promoter in all three cases. Other determinations (supercoil relaxation, T7 gene 3 product sensitivity, bromoacetaldehyde reactivity, anomalous gel mobility, the influence of negative superhelical density on nuclease sensitivity) led to the conclusion that the B-helix deformation was not due to a previously recognized DNA conformation (left-handed Z-DNA, cruciform, bent DNA), but may be accounted for by the homopurine X homopyrimidine nature of this region.     

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.     

Positive and negative regulatory DNA elements including a CCArGG box are involved in the cell type-specific expression of the human muscle dystrophin gene.

The muscle-specific promoter of the dystrophin gene is active in skeletal, cardiac, and smooth muscles and is specifically stimulated during differentiation of myoblasts into multinucleated myotubes. An 850-base pair (bp) DNA fragment upstream from the cap site is able to confer a partial muscle specificity to a reporter gene. The region between -850 and -140 bp includes nonspecific negative and positive regulatory sequences. A continuous stretch of 140 bp upstream from the cap site exhibits a striking conservation between rodents and human (93% homology) and still retains muscle preference of expression. It contains two putative binding sites for factors involved in regulation of other muscle-specific genes, a CCArGG box and an E box. This latter element, however, is unable to confer the ability to be transactivated by MyoD1 to the dystrophin promoter. The -140-bp promoter fragment exhibits antagonist effects contributed by one inhibiting sequence (nucleotide -140/-96), active in all cell types, and one activating region, from nucleotide -96 to the cap site, sufficient to confer a muscle preference of expression, in which the CCArGG box seems to play a major role.     

灰色链霉菌启动子活性片段在大肠杆菌中的亚克隆及其序列分析

通过对重组质粒No.8-1的亚克隆,灰色链霉菌插入到大肠杆菌载体质粒中的序列已缩小到410bp,仍具有启动子功能。序列分析表明,启动子活性片段的G C碱基组成为50.5%。内含链霉菌启动子区域常有的正向重复序列;有1个Alul位点,1个Clal位点,2个Mbol位点,3个Nla Ⅲ位点,1个Pvu Ⅱ位点;具有类似于E.coli启动子的保守序列-10区和-35区,两者间隔18bp;在相应位置上分别有一段序列与E.coli的SD序列和在苄铅青链霉菌的SEP(Streptomyces-E.coil-type promoter)序列中存在的保守序列具有一定的相似性。     

Visualization of the bent helix in kinetoplast DNA by electron microscopy

Kinetoplast DNA minicircles from the trypanosomatid Crithidia fasciculata contain a segment of approximately 200 bp which is probably more highly bent than any other DNA previously studied. Electron microscopy (EM) of relaxed minicircles (2.5 kb) revealed 200-300 bp loops within the larger circles, and the loops could also be detected on full-length linear molecules. Examination by EM of a 219 bp cloned fragment which contains the bent helix revealed that up to 70% of the molecules appeared circular whether or not the ends were cohesive. In contrast, a 207 bp fragment from pBR322 showed no circles and the fragments in general appeared much straighter than the kinetoplast fragments. Treatment of the 219 bp bent kinetoplast fragment with the drug distamycin caused a striking reduction in curvature.