Interaction Between HMG Proteins (1+2) and the Negative Regulatory Region 1(NCR1) in the 5''''-flanking Sequence of the Human β-globin Gene

The pattern of high mobility group proteins 1 and 2 (HMG1,2) interaction with the 5'-flanking sequence of the human β-globin gene has been analyzed by scanning tunnelling microscopy (STM). A 200 bp negative regulatory region in the 5'-flanking sequence of the human β-globin gene can be folded by HMG proteins 1 and 2 into a circular structure (diameter 70±6) with a linear tail which seems to be a left-handed double helix structure.     

酵母丙氨酸转移核糖核酸3′-半分子(36—76)的合成

本文报告用T_4RNA连接酶将相应于酵母丙氨酸转移核糖核酸(tRNA_y~(Ala))3′-半分子(36—76)的三个寡核昔酸大片段——10(36—45)(Ⅰ),12(46—57)(Ⅱ)和19p(58—76)(Ⅲ)——从3′-端向5′-端延伸逐个连接合成了这个tRNA的3′-半分子(36—76)。首先在供受体配比为1:1.5的情况下,采取三步连续反应,即19p(Ⅲ)的5′-磷酸化,然后与12(Ⅱ)的连接和连接反应产物的5′-磷酸化等反应,一次制备分离的方法,以70％的总得率合成了5′-磷酸化的三十一核苷酸(46—76)(Ⅳ)。然后,以(Ⅳ)作为下一步反应的供体和三倍量的10(Ⅰ)连接,以67％的产率合成了具有四十一核苷酸的tRNA_y~(Ala)的3′-半分子(36—76)(Ⅴ)。将这个合成的3′-半分子,5′-磷酸化以后,与天然的5′-半分子连接,人工半合成了tRNA_y~(Ala)整分子,经生物活力测定,这个人工半合成的tRNA_y~(Ala)具有接受[~3H]-丙氨酸、并能将接受的丙氨酸转移到蛋白质分子中去的生物活力。     

Binding of HMG Proteins to the 5''''_Flanking Sequence of Human β-Globin Gene

Our previous studies have identified that there are at least three regulatory regions (two negative regions and one positive region) in the 5'-flanking sequence of human β-globin gene (-610 to +1 bp). The binding of HMG proteins to both negative regulatory regions was examined by the gel mobility shift and DNase I protection assays.In gel mobility shift assay,we observed that HMG proteins 1 and 2 could bind to both negative regulatory regions (NCR1 and NCR2).Using the gel shift competition assay,we identified that the binding proteins between the two regions are different from each other.DNase I protection analysis shows that HMG proteins 1 and 2 only bind to one site (between-560 and-533 bp) in NCR1.However,two protected regions can be detected in NCR2, one between-272 and-252 bp relative to the cap site, the other between-306 and-329 bp.We also observed that HMG proteins 14 and 17 could not bind to both negative regions, so it seems that HMG proteins 1 and 2 may play an important role in the regulati     

人体β-珠蛋白基因5′-旁侧DNA序列内的负调控区域1(NCR1)与HMG蛋白质(1+2)的相互作用

利用分子生物学技术和扫描隧道显微镜方法首次观察到HMG蛋白质(1+2)和人体β-珠蛋白基因的5′-旁侧DNA序列内的一个负调控区域(NCR1)相互作用的构象,这类蛋白质能将线状的DNA片段(～200bp)折叠起来,形成一个环状结构(直径70±6A)和°一条似乎呈现左手螺旋(z-from)的线状DNA尾巴。     

HMG蛋白质与人体β-珠蛋白基因5旁侧DNA序列的结合

人体β-珠蛋白基因的5′-旁侧DNA片段(-610bp→+1bp)内的两个负调控区域与HMG蛋白质(High Mobility Group Proteins)结合状况已用近代分子生物学的技术(足印分析法(DNase Ⅰ protection Assay)和凝胶电泳阻抑分析法(Gel MobilityShift Assays)加以分析。用凝胶电泳阻抑分析方法证明了HMG蛋白质(1+2)与两个负调控区域有不同程度的结合,同时也观察到这两个负调控区域的结合蛋白是各异的。为了进一步检测HMG蛋白质(1+2)与DNA序列的精确结合位点,我们又采用了足印分析的方法,观察到在第一个负调控区内HMG蛋白质(1+2)有一个专一的结合位点(-560bp→-533bp)。在第二个负调控区内,则可以检测到有两个结合位点(-272bp→-252bp和-306bp→-329bp)。另外,我们还证实了HMG蛋白质(14+17)与这两个负调控区都不能结合。上述实验结果提示HMG蛋白质(1+2)在β-珠蛋白基因表达中起着积极的调控作用。     

SYNTHESIS OF THE 3'-HALF MOLECULE OF YEAST ALANINE tRNA

This paper deals with the synthesis of the 3'-half molecule of yeast alanine transfer RNA (tRNA_y~(Ala)) by ligation with T_4 RNA ligase of three component oligonucleotide fragments corresponding to nucleotides 36-45(Ⅰ), 46-57(Ⅱ) and 58-76(Ⅲ) in succession extending from the 3'-end to the 5'-end. First, in a ratio of acceptor to donor at 1.5 to 1, we adopted a method of three successive reactions, namely, the 5'-phosphorylation of the nonadecamer (Ⅲ), ligation with the dodecamer(Ⅱ) and the 5'-phosphorylation of the ligation product formed; with one isolation step and obtained the 5'-phosphorylated 31mer(46-76) (Ⅳ) in an overall yield of 70%. Then the 31met(Ⅵ) as a donor was ligated with 3 times of decamer(Ⅰ) to form the 41met(36-76) (Ⅴ), the 3'-half molecule of tRNA_y~(Ala)). The yield was 67%. After 5'-phosphorylation, (Ⅴ) was ligated with the natural 5'-half molecule to form the semi-synthetic tRNA_y~(Ala)), which was biologically active, i. e. accepting and transferring (~3H)-alanine into p