酵母基因内含子中转录正调控位点的统计分析

以一组随机选取的对照序列中寡核苷酸的出现频率为基础,分析转录频率较高的酵母基因内含子序列的寡核苷酸使用情况,抽提出一批过表达的寡核苷酸。然后对抽取出的寡核苷酸和它们在每个高效转录基因内含子序列中重叠、连接后形成的长寡核苷酸序列片段进行分析．并对照实验结果和用比较分析方法获得的结果、推测这些寡核苷酸可能是高效转录基因内含子序列中潜在的转录因子结合位点。     

THE USE AND EVALUATION OF 2+2 PHOTOADDITION IN IMMOBILIZATION OF OLIGONUCLEOTIDES ON A THREE DIMENSIONAL HYDROGEL MATRIX

Photochemical attachment of synthetic oligonucleotides on the three dimensional surface of a polyacrylamide based hydrogel was used in the specific detection of target oligonucleotides. Covalent attachment of the oligonucleotide to the hydrogel was mediated by the incorporation of a 2+2 photo-attachable functional group in both the hydrogel and the oligonucleotide probe. Expression and SNP assays were used to evaluate this platform.     

ANTIVIRAL ACTIVITY OF STERIC-BLOCK OLIGONUCLEOTIDES TARGETING THE HIV-1 TRANS-ACTIVATION RESPONSE AND PACKAGING SIGNAL STEM-LOOP RNAS

Mixmer oligonucleotides consisting of residues of both 2′-O-methylnucleosides (OMe) and locked nucleic acids (LNA) were designed targeting two stem-loops in the 5′-UTR of HIV-1 RNA, the trans-activation response region (TAR), which is the site of binding of the Tat protein, and the SL3 loop, which is the primary packaging element that binds the Gag polyprotein. These oligonucleotides were found to inhibit syncitia formation dose- and sequence-dependently when delivered to HeLa T4 LTR β-Gal cells and subsequently infected with HIV-1.     

鞘内注射M受体和GDNF反义寡脱氧核苷酸抑制吗啡戒断大鼠蓝斑c-Fos表达

应用免疫组化方法观察鞘内注射毒蕈碱型乙酰胆碱(muscarinic acetylcholine receptor,M) 受体和胶质细胞源性神经营养因子(glial cell derived neurotrophic factor,GDNF)反义寡脱氧核苷酸对吗啡戒断大鼠蓝斑(locus coeruleus,LC)区内Fos表达的影响。结果显示,鞘内注射M_2受体和GDNF反义寡脱氧核苷酸明显减少大鼠吗啡戒断症状评分值(n=6,P<0.05)。正常大鼠LC区神经元Fos基础表达较低,吗啡依赖大鼠LC区神经元Fos表达增加,吗啡依赖大鼠纳酪酮(4mg/Kg,ip)催促戒断后,Fos表达进一步增加;鞘内注射M_2受体和GDNF反义寡脱氧核苷酸处理后均减少吗啡戒断大鼠LC区神经元Fos表达(n=5,P<0.05)。而鞘内注射M_1受体反义寡脱氧核苷酸处理组LC 区神经元Fos表达较吗啡戒断组没有显著差异(n=5,P>O.05)。结果提示:脊髓M_2受体调节吗啡戒断时LC区的神经元激活,而这种神经上行性激活涉及神经元与胶质细胞之间的适应性调节。     

FISH检测快速诊断曲霉菌感染的临床价值

目的探讨荧光原位杂交技术(FISH)检测曲霉菌感染的可行性和特异性,以期早期病原学诊断临床真菌感染。方法采用地高辛末端标记的18S-1寡核苷酸探针与27例经HE及PAS染色诊断为可疑真菌感染的石蜡包埋组织切片进行FISH检测。结果 HE及PAS染色可疑真菌感染分别为23例和27例。27例可疑真菌感染标本14例检出FISH阳性信号。结果 FISH可以特异性地检测出组织石蜡切片中的曲霉菌,设计不同菌属探针进行靶DNA杂交可以快速检测各种临床标本中的真菌感染,从而作为IFI早期病原学诊断的一种新策略。     

THE SYNTHESIS OF,AND STUDIES ON, 2′-C-MODIFIED NUCLEOTIDES

The synthesis of uridine monomers containing either a 2′-deoxy-2′-C-methy- lcyano or ethylcyano group is described. These monomers are intended for incorporation into oligonucleotides to investigate a proposed duplex-stabilising effect exerted by 2′-tethered amide groups.     

A NOVEL APPROACH FOR THE SOLID PHASE SYNTHESIS OF DNA-PEPTIDE CONJUGATES

The strategy of this study involves automated synthesis of oligonucleotides on a CPG support using standard cyanoethyl phosphoramidite chemistry followed by covalent linkage to peptide fragments bearing a free terminal α-amino group and residues with protected side chains. Conjugation was formed through an alkyldiisocyanate linker. Conjugates wereisolated by cleavage from the solid support and deprotection in one step.     

A COMPARATIVE STUDY OF SUPPORTS FOR THE SYNTHESIS OF OLIGONUCLEOTIDES WITHOUT USING AMMONIA

Abstract

A comparative study of the cleavage efficiency of succinyl, phthaloyl, oxalyl, 2-(2-nitrophenyl)ethyl, 9-fluorenylmethyl, and 2-nitrobenzyl supports in 0.5M DBU solutions is described. A decrease in cleavage efficiency is observed when small oligonucleotides containing thymidine are linked to the supports. In these conditions oxalyl supports gave the best yields followed by 2-(2-nitrophenyl)ethyl and 9-fluorenylmethyl supports.     

阳性脂质体介导反义寡聚核苷酸转染HeLa细胞的效果研究

采用FITC标记的未经修饰的和经过修饰的两种19-mer反义寡聚核苷酸序列(ODN19和S-ODN19)作为转染物质,用流式细胞技术(FCM)研究比较几种常用阳性脂质体介导的寡聚核苷酸转染HeLa细胞的效果及适宜的转染时间。未经化学修饰的ODN19转染结果显示,LipofectAmine和DM-RIE-C增强转染的作用相对较强,而其他两种脂质体的作用并不明显。对于经过修饰的S-ODN19转染而言,四种阳性脂质体均具有增强S-ODN19转染作用,但以LipofectAmine的效果最为明显,其转染效果(FITC均值为5203.11)为无脂质体介导对照的数十倍。四种阳性脂质体的增强转染作用排序为:LipofectAmine>FuGENE6>Lipofectin>DM-RIR-C。另外,在用FuGENE6介导寡聚核苷酸转染时,采用4小时转染时间可获较好转染效果。     

COVALENT MODIFICATION AND SURFACE IMMOBILIZATION OF NUCLEIC ACIDS VIA THE DIELS-ALDER BIOCONJUGATION METHOD

The importance of chemically modified and surface immobilized nucleic acids has inspired the development of a wide variety of complementary techniques for covalent oligonucleotide preparation and immobilization. We are developing technology based on the use of a Diels-Alder reaction for accomplishing the covalent modification of oligonucleotides. Reported herein is preliminary progress toward the establishment of robust reagents for introducing the reactive functionality, as well as studies employing the BIACORE system to demonstrate surface immobilization by the method.     

A NEW CLASS OF ANTI-HIV-1 OLIGONUCLEOTIDE TARGETED TO THE POLYPURINE TRACT OF VIRAL RNA

The PPT is highly conserved among the known HIV-1 strains, and is a possible target for triplex formation. We show triple-helix formation by a two-strand-system (FTFOs, DsDGloopT5-37) targeted to the polypurine tract (PPT) of HIV-1. In HIV-1 infected MOLT-4 cells, the FTFOs containing phosphorothioate groups at the antisense strand and guanosine rich parts within the third Hoogsteen base pairing sequence inhibit the replication of HIV-1 more effectively than the antisense phos-phorothioate oligonucleotides indicating sequence-specific inhibition of HIV-1 replication for 62 days. However, AZT, treated cells expressed high levels of p 24 products after 46 days.     

RECOGNITION OF DNA BY STRAND INVASION WITH OLIGONUCLEOTIDE-SPERMINE CONJUGATES

Modified oligonucleotides bearing spermine groups (ODN-sper) with increased binding affinity to DNA have been synthesized. The ability of these ODN-sper to bind within superhelical double-stranded DNA by strand invasion has been studied. The uptake by a supercoiled plasmid was 3 fold higher for the ODN-sper than for the unmodified oligonucleotides.     

几株球形芽孢杆菌二元毒素基因的检测及对敏感和抗性尖音库蚊的毒性

根据Bacillus sphaericus 2362二元毒素基因核苷酸序列合成的一组寡聚核苷酸为引物,通过PCR扩增出1.1 kb的DNA片段作探针检测了C₃-41、IAB881、IAB872、BS-197和lPl-G菌株中二元毒素基因。Southern杂交表明C₃-41、IAB881、IAB872和BS-197菌株中3.5kb Hind III及LPl-G中4.7kb Hind III的酶切片段分别带有与探针有高度同源性的二元毒素基因。SDS-PAGE和Western印迹杂交表明所有菌株都能产生41.9kD和51.4kD的毒素蛋白。C₃.41、IAB881、BS.197和2362的全发酵液和碱抽提液对敏感尖音库蚊Culex pipienssubsp.Pipiens幼虫毒性高,但对抗性幼虫几乎无毒,LPl-G对敏感和抗性蚊幼虫具有相同的中度毒杀作用;IAB872对敏感幼虫毒性高,对抗性幼虫的毒力同LPl-G相似。这两株菌对抗性蚊幼虫的毒性可能是由Mtx毒素蛋白所导致的。     

SIGNIFICANT IMPROVEMENT OF QUALITY FOR LONG OLIGONUCLEOTIDES BY USING CONTROLLED PORE GLASS WITH LARGE PORES

A key factor influencing the quality of long oligonucleotides is the choice of controlled pore glass (CPG) which is used as a solid support during oligonucleotide synthesis. We studied the influence of CPG pore size on the quality of 75-mer oligonucleotides. Using electrophoresis and HPLC, we demonstrated failure modes that can occur at certain oligo lengths with 1000A pore size, and compared yield and purity of 75-mer oligos using 1000A and larger pore size CPG. We showed that oligonucleotides with much better quality are obtained using CPG with pore sizes of 1400A and larger. We also identified the key characteristics for CPG selection that lead to the best CPG performance.     

中国家蚕抗菌肽ABP-CM4在E.coli中的融合表达及活性分析

参照天然抗菌肽CM4(ABP-CM4)氨基酸序列和大肠杆菌偏爱密码子,采用rPCR法获得CM4基因后重组到表达载体pET32a上,在E.coli中融合表达。表达产物以可溶性存在,经Ni2 -NTA琼脂糖亲和层析获得融合蛋白,再经甲酸切割、亲和层析和阳离子交换层析,得到纯化的重组抗菌肽。琼脂糖扩散法和液相测定法证明了纯化的抗菌肽具有抗菌活性。     

THE γ SUBUNITS OF PHYCOERYTHRIN FROM A RED ALGA: POSITION IN PHYCOBILISOMES AND SEQUENCE CHARACTERIZATION

Aglaothamnion neglectum Feldman-Mazoyer has two γ subunits, γ³¹ and γ³³, that are associated with phycoerythrin in the light-harvesting phycobilisomes. We demonstrate that these subunits are spatially separated within the phycobilisome, with the γ³¹ subunit present at the distal end of phycobilisome rods and the γ³³ subunit present on the proximal end. These subunits are thought to link phycoerythrin hexamers together in the rod substructure, serving a role analogous to that of linker polypeptides of cyanobacteria (although unlike the cyanobacterial linker polypeptides they are chromophorylated). The sequencing of tryptic polypeptides of the γ subunits enabled us to prepare oligonucleotides encoding different regions of γ³¹. These oligonucleotides were used as primers to generate a probe for isolating a γ³¹ cDNA clone. Characterization of the cDNA clone predicts a polypeptide of 280 amino acids with a 42 amino acid presequence that is characteristic of a transit peptide, the peptide that targets proteins to chloroplasts of vascular plants. The γ³¹ subunit has 50% similarity to the previously characterized γ³³ subunit but has no identifiable similarity to functionally related polypeptides present in cyanobacterial phycobilisomes or to any other polypeptides in the databases. A repeat of 95 amino acids is present in the red algal γ subunit sequences, suggesting that these proteins were generated by a gene duplication followed by fusion of the duplicate sequences.     

PREPARATION OF OLIGODEOXYNUCLEOTIDE 5′-TRIPHOSPHATES USING SOLID SUPPORT APPROACH

We report a synthetic procedure for conversion of oligonucleotides to their 5′-triphosphate derivatives with moderate yield. The oligonucleotides were synthesized on solid support using standard phosphoramidite protocols. The DMT protection group was removed and the 5′-OH was phosphitylated using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one followed by reaction with tributyammonium pyrophosphate and iodine oxidation. After subsequent removal from support and complete deprotection, the products were isolated by anion-exchange HPLC chromatography. Structures of several 5′-triphosphate derivatives have been proven by phosphorus NMR, Mass-spectrometry and by HPLC comparison with authentic samples.     

抗芽殖酵母端粒G4-DNA结构的单克隆抗体和基因工程单链抗体片段的制备和定性研究

大多数真核生物端粒3'末端由富含鸟苷酸的重复序列组成,并可以在体外形成四链G4- DNA结构。为了解这种结构是否在体内存在,本文中我们以芽殖酵母作为研究对象,将G4-DNA作为抗原免疫BALB／c小鼠,制备抗G4-DNA的单克隆抗体,结果显示该抗体能够特异性识别富含鸟苷酸重复序列DNA。为了提高抗体的特异性,我们通过基因工程制备抗体:利用RT-PCR的方法,得到抗体重链和轻链可变区的基因,然后克隆到载体pET22b中得到表达质粒pET22b-scFv,转入大肠杆菌进行表达。在细胞周质中我们检测并纯化到了目的基因的表达产物。另外,我们还利用该基因序列进行了初步的结构分析。基因工程抗体在大肠杆菌中的成功表达及结构分析为今后利用该抗体进行定点突变来研制高特异性和亲和力的抗G4-DNA抗体奠定了基础。     

PHOSPHONOACETATE DERIVATIVES OF OLIGODEOXYRIBONUCLEOTIDES

Abstract

A deoxyribodinucleotide phosphonoacetate derivative has been prepared, separated into individual diastereomers, and incorporated into oligodeoxyribonucleotides possessing alternating phosphodiester and phosphonoacetate backbone linkages. The hybridization properties and enzymatic stabilities of these oligonucleotides have been studied.