Isolation and cloning of putative mouse DNA replication initiation sites: binding to nuclear protein factors.

By using an original two-step technique (trioxsalen crosslinking/immunoprecipitation) we were able to isolate in a single-stranded form a fraction of mouse DNA enriched in putative Replication Initiation Sequences (RIS). The isolated and purified single-strand fragments were made double-stranded in vitro and were cloned in pUC12 to prepare a confined RIS library. 30 randomly selected RIS inserts were subjected to gel mobility shift assay using nuclear extracts either from dividing, or from quiescent mouse cells. Twelve out of the 30 RIS fragments showed specific binding to proteins present in nuclear extract from dividing cells, while none were retarded by extracts from quiescent cells. RIS12, RIS18 and RIS30 were sequenced and it was found that they were A+T rich and contained different regulatory elements. By using a two step procedure (Heparin-sepharose chromatography/DNA affinity chromatography) we isolated the protein factor that specifically binds to RIS12. It appeared as a double band with apparent molecular masses of 63 and 65 kD.     

免疫测定光敏核不育水稻中光敏色素Ⅰ含量

光敏核不育水稻（农垦５８Ｓ）是中国水稻研究中的一个重要发现,在水稻杂交育种上有巨大潜力。研究表明：农垦５８Ｓ的雄性不育性受光周期调控,光敏色素是育性转变过程中光周期反应的光受体,然而,鉴于高等植物中存在至少两种不同的光敏色素分子,并且它们同时存在于水稻中,人们对调节农垦５８Ｓ雄性不育过程的光敏色素分子种类及作用方式仍然不清楚。本文采用酶联免疫吸附测定法（ＥＬＩＳＡ）,比较了不同光周期下农垦５８Ｓ和对照品种农垦５８叶片（光周期感受器官）中光敏色素Ｉ（ｐｈｙＡ）的含量。从农垦５８Ｓ的二次枝梗原基分化期开始,进行１０天的光周期处理。一组为短日照（ＳＤ）,另一组为长日照（ＬＤ）。在第１０个暗期结束前,于暗绿光下收获每株水稻的最上部两片新展叶,立即保存在液氮中。样品在研钵中磨成液氮粉,然后加入提取液（含５０ｍｍｏｌ／ＬＴｒｉｓ－ＨＣＩ和０．２ｍｏｌ／Ｌ巯基乙醇,ｐＨ８．５）匀浆。粗提液经０．５％聚乙烯亚胺（ＰＥＩ）沉淀后,上清液供ＥＬＩＳＡ分析。以燕麦ｐｈｙＡ的多克隆抗体、单克隆抗体分别作ＥＬＩＳＡ的一抗和检测抗体。采用ＥＬＩＳＡ可以专一性地检测水稻ｐｈｙＡ（Ｔａｂｌｅ１）。几次独立的实验说明,光周期处理对ｐｈｙＡ含     

Regulation of alpha-amylase-encoding gene expression in germinating seeds and cultured cells of rice.

Four alpha-amylase-encoding cDNA (alpha Amy-C) clones were isolated from a cDNA library derived from poly(A)+RNA of gibberellic acid (GA3)-treated rice aleurone layers. Nucleotide sequence analysis indicates that the four cDNAs were derived from different alpha Amy genes. Expression of the individual alpha Amy gene in germinating seeds and cultured suspension cells of rice was studied using gene-specific probes. In germinating seeds, expression of the alpha Amy genes is positively regulated by GA3 in a temporally coordinated but quantitatively distinct manner. In cultured suspension cells, in contrast, expression of the alpha Amy genes is negatively and differentially regulated by sugars present in the medium. In addition, one strong and one weak carbohydrate-starvation-responsive alpha Amy genes have been identified. Interactions between the promoter region (HS501) of a rice alpha Amy gene and GA3-inducible DNA binding proteins in rice aleurone cells were also studied. A DNA mobility-shift assay showed that the aleurone proteins interact with two specific DNA fragments within HS501. One fragment is located between nt -131 to -170 and contains two imperfect directly repeated pyrimidine elements and a putative GA3-response element. The other fragment is located between nt -92 to -130 that contains a putative enhancer sequence. The interactions between aleurone proteins and these two fragments are sequence-specific and GA-responsive.     

Fertility response to photoperiod and temperature in indica photoperiod-sensitive male sterile rice

The aim of this work was to develop a photoperiod-sensitive male sterile rice with stable sterility. We developed Changguang S, an indica rice strain, by using a short critical day length. Differences in the fertility responses of Changguang S strain pollen to temperature and photoperiod under natural and controlled conditions were studied. The results showed that Changguang S strain exhibited stable sterility under long-day and low-temperature conditions (22°C, 15 days). The stability of sterility was significantly higher than that of other such rice strains, Nongken 58S and 7001S. The critical photoperiod for inducing male sterility in Changguang S was 13 h or shorter, and its duration was significantly shorter than that required for rice strains Nongken 58S and 7001S. It is suggested that Changguang S is a typical photoperiod-sensitive male sterile rice strain with a shorter critical day length and a lower critical temperature. It is promising to apply this strain to two-line hybrid rice production.     

Differential binding of a CCAAT DNA binding factor to the promoters of the mouse alpha 2(I) and alpha 1(III) collagen genes

An exonuclease III assay (Wu, C. (1985) Nature 317, 84-87) was used to identify in nuclear extracts of NIH 3T3 cells a factor which binds to the CCAAT segment of the alpha 2(I) collagen promoter between -80 and -84. This sequence is located on the coding strand in the alpha 2(I) collagen promoter. Binding is specific since only promoter fragments which contain the CCAAT box sequences on one or the other DNA strand inhibit binding to the alpha 2(I) collagen CCAAT box. The CCAAT binding factor protects approximately 26 base pairs of the alpha 2(I) collagen promoter from exonuclease III digestion. Binding to the alpha 2(I) collagen promoter CCAAT box is not inhibited by a fragment of the alpha 1(III) collagen promoter (from -396 to +16), which does not contain a CCAAT sequence on either one or the other strand. Our data suggest that two genes such as the alpha 2(I) and alpha 1(III) collagen genes, which are coordinately expressed in many tissues, are not necessarily regulated by the same trans-acting DNA binding proteins.     

AchR亚基基因启动子与分化/未分化肌细胞核因子的相互作用分析

采用凝胶阻滞实验比较分析了鸡烟碱样乙酰胆碱受体（ＡＣｈＲ）亚基基因－２７５／＋３６片段与分化／未分化肌细胞核抽提物的相互作用．发现未分化肌细胞核内存在两种直接识别ＡＣｈＲ启动子的结合活性,其结合反应不能被ＡＣｈＲα亚基基因增强子（含Ｅ盒子）竞争阻断,揭示此结合活性与ＭｙｏＤ家族无关,并表现为基因特异性结合;两种结合活性中一种结合活性既存在于未分化细胞,也存在于分化肌细胞,另一种结合活性只存在于未分化肌细胞．存在于未分化肌细胞、特异识别基因的结合活性不同于ＭｙｏＤ家族,也不同于已发现的Ｉｄ和Ｉ－ｍｆ（两者不能直接结合ＤＮＡ）,可能与基因在未分化肌细胞中表达的负调控有关．     

Rice proteins that bind single-stranded G-rich telomere DNA

In this work, we have identified and characterized proteins in rice nuclear extracts that specifically bind the single-stranded G-rich telomere sequence. Three types of specific DNA-protein complexes (I, II, and III) were identified by gel retardation assays using synthetic telomere substrates consisting of two or more single-stranded TTTAGGG repeats and rice nuclear extracts. Since each complex has a unique biochemical property and differs in electrophoretic mobility, at least three different proteins interact with the G-rich telomere sequences. These proteins are called rice G-rich telomere binding protein (RGBP) and none of them show binding affinity to double-stranded telomere repeats or single-stranded C-rich sequence. Changing one or two G's to C's in the TTTAGGG repeats abolishes binding activity. RGBPs have a greatly reduced affinity for human and Tetrahymena telomeric sequence and do not efficiently bind the cognate G-rich telomere RNA sequence UUUAGGG. Like other telomere binding proteins, RGBPs are resistant to high salt concentrations. RNase sensitivity of the DNA-protein interactions was tested to investigate whether an RNA component mediates the telomeric DNA-protein interaction. In this assay, we observed a novel complex (complex III) in gel retardation assays which did not alter the mobilities or the band intensities of the two pre-existing complexes (I and II). The complex III, in addition to binding to telomeric sequences, has a binding affinity to rice nuclear RNA, whereas two other complexes have a binding affinity to only single-stranded G-rich telomere DNA. Taken together, these studies suggest that RGBPs are new types of telomere-binding proteins that bind in vitro to single-stranded G-rich telomere DNA in the angiosperms.