Development of a FIA system with immobilized enzymes for specific post-column detection of purine bases and their nucleosides separated by HPLC column.

A sensitive and highly selective method for the simultaneous determination of purine bases and their nucleosides is proposed. An amperometric flow-injection system with the two immobilized enzyme reactors (guanase immobilized reactor and purine nucleoside phosphorylase/xanthine oxidase co-immobilized reactor) is used as the specific post-column detection system of HPLC, to convert compounds separated by a reversed-phase. HPLC column to electroactive species (hydrogen peroxide and uric acid) which can be detected at a flow-through platinum electrode. The proposed detection system is specific for a group of purine bases and purine nucleosides and does not respond for purine nucleotides and pyrimidine bases. The linear determination ranges are from 10 pmol to 5 nmol for four purine bases (hypoxanthine, xanthine, guanine, and adenine) and four purine nucleosides (inosine, xanthosine, guanosine, and adenosine). The detection limits are 1.2-5.5 pmol.     

白百利烟草愈伤组织细胞细胞分裂素结合蛋白的研究

利用BA-Sepharose 4B亲和层析技术丛白百利烟草(Nicotiana tabacum Baibaili)愈伤组织细胞分离提纯了分子量为4400±100道尔顿的细胞分裂素结合蛋白(CB-蛋白)。在细胞表面、核糖体、线粒体、叶绿体和细胞核上以及在细胞液中都有CB-蛋白存在,而核糖体上的CB-蛋白含量量高。探讨了CB-蛋白的功能。     

小鼠早期胚胎发育期间TGF-β_1免疫组织化学定位

用兔抗人血小板TGF-β_1 N末端1—29氨基酸残基人工合成多肽抗血清作探针以及免疫荧光和免疫酶染色技术,分析了1—12天小鼠早期发育期间胚胎的TGF-β_1物质分布。结果表明,着床前胚胎包括卵裂细胞,桑椹胚和胚泡的ICM及滋养外胚层等细胞均显示TGF-β_1阳性免疫荧光染色。免疫酶染色还证明,沿囊胚腔顶部单层排列的原始内胚层细胞比邻近的ICM细胞有较深的染色反应。随着胚胎着床和进一步发育,7天龄胚胎中胚层早期形成阶段,紧靠中胚层一侧的外胚层胞质中含有浓集的棕色颗粒;各胚层的部分区域也存在着染色强度上差别。8—12天龄胚胎中,体节,心壁、间质细胞和肠道以及卵黄囊的脏壁中胚层均有显著的TGF-β_1免疫酶阳性物质。这些结果表明,着床前小鼠胚胎富含TGF-β_1物质,着床后的胚外组织,例如卵黄囊也为胚胎进一步发育提供了富含TGF-β_1物质的微环境;同时也提示,小鼠早期胚胎发育期间的胚泡形成,ICM细胞分化出原始内胚层,卵柱期中胚层形成,以及以后的神经管、体节和肢芽形成阶段等一系列形态发生和器官形成过程中,TGF-β_1可能是参与重要作用的一种生长调节因子。     

人体基因组随机单拷贝顺序的分离及其在染色体上的定位

本文从构建杂种细胞14-7-1的基因组文库出发,用种特异的探针分离出含有人体基因组顺序的重组子,并进一步分析了其中13个克隆,得到8个单拷贝顺序。通过与已建立的杂种细胞克隆分布板杂交以及染色体的原位杂交方法,将1个单拷贝顺序FD11-1定位在11p11-q11上。由于已经报道在11号染色体上具有3个连锁群,它们分别位于11p15、11p13和11q13上,因此,FD11-1有可能为11号染色体连锁基因图的建立提供1个有意义的座位。     

New telomere formation coupled with site-specific chromosome breakage in Tetrahymena thermophila.

Programmed chromosome breakage occurs in many ciliated protozoa and is accompanied by efficient new telomere formation. In this study, we have investigated the relationship between programmed chromosome breakage and telomere formation in Tetrahymena thermophila. Using specially constructed DNA clones containing the breakage signal Cbs in transformation studies, we have determined the locations of telomere addition around the breakage sites. They occur at variable positions, over 90% of which are within a small region (less than 30 bp) starting 4 bp from Cbs. This distribution is independent of the nucleotide sequence in the region or of the orientation of Cbs. In five of six cases determined, these sites occur at or before a T, and in the remaining case, the site occurs at or before a G. When sequences devoid of G or T are placed in this region, telomere addition still occurs within the region to maintain a similar distance relationship with Cbs. This efficient and healing process appears to be associated specifically with Cbs-directed breakage, since it does not occur when DNA ends are generated by restriction enzyme digestion. These results suggest a strong mechanistic link between chromosome breakage and telomere formation.     

Sequence Variations in the Bovine Growth Hormone Gene Characterized by Single-Strand Conformation Polymorphism (Sscp) Analysis and Their Association with Milk Production Traits in Holsteins

Sequence variations in the bovine growth hormone (GH) gene were investigated by single strand conformation polymorphism (SSCP) analysis of seven amplified fragments covering almost the entire gene (2.7 kb). SSCPs were detected in four of these fragments and a total of six polymorphisms were found in a sample of 128 Holstein bulls. Two polymorphisms, a T->C transition in the third intron (designated GH4.1) and an A->C transversion in the fifth exon (designated GH6.2), were shown to be associated with milk production traits. GH4.1(c)/GH4.1(c) bulls had higher milk yield than GH4.1(c)/GH4.1(t) (P <= 0.005) and GH4.1(t)/GH4.1(t) (P <= 0.0022) bulls. GH4.1(c)/GH4.1(c) bulls had higher kg fat (P <= 0.0076) and protein (P <= 0.0018) than GH4.1(c)/GH4.1(t) bulls. Similar effects on milk production traits with the GH6.2 polymorphism were observed with the GH6.2(a) allele being the favorable allele. The average effects of the gene substitution for GH4.1 and GH6.2 are similar, with +/-300 kg for milk yield, +/-8 kg for fat content and +/-7 kg for protein content per lactation. The positive association of GH4.1(c) and GH6.2(a) with milk production traits may be useful for improving milk performance in dairy cattle.     

Evolutionary Conservation of Sequences Directing Chromosome Breakage and Rdna Palindrome Formation in Tetrahymenine Ciliates

Extensive, programmed chromosome breakage occurs during formation of the somatic macronucleus of ciliated protozoa. The cis-acting signal directing breakage has been most rigorously defined in Tetrahymena thermophila, where it consists of a 15-bp DNA sequence known as Cbs, for chromosome breakage sequence. We have identified sequences identical or nearly identical to the T. thermophila Cbs at sites of breakage flanking the germline micronuclear rDNA locus of six additional species of Tetrahymena as well as members of two related genera. Other general features of the breakage site are also conserved, but surprisingly, the orientation and number of copies of Cbs are not always conserved, suggesting the occurrence of germline rearrangement events over evolutionary time. At one end of the T. thermophila micronuclear rDNA locus, a pair of short inverted repeats adjacent to Cbs directs the formation of a giant palindromic molecule. We have examined the corresponding sequences from two other Tetrahymena species. We find the sequence to be partially conserved, as previously implied from analysis of macronuclear rDNA, but of variable length and organization.     

肿瘤坏死因子－α（TNF－α）对常见呼吸道病毒的抑制作用

用人重组肿瘤坏死因子－α（Ｔｕｍｏｒｎｅｃｒｏｓｉｓｆａｃｔｏｒ－α,ＴＮＦ－α）和人天然α干扰素（Ｉｎｔｅｒｆｅｒｏｎ－α－,ＩＦＮ－α）在人胚胎肺纤维母细胞（ＨＥＦ）和Ｈｅｐ－２细胞系上对常见呼吸道病毒所致细胞病变抑制进行比较观察。病毒包括不同型别的腺病毒５株,单疱病毒Ⅰ型（ＨＳＶ－Ｉ）１株,鼻病毒１株,仙台病毒１株,ＶＳＶ１株。结果提示ＴＮＦ－α和ＩＦＮ－α均具有广谱抗病毒活性。ＴＮＦ－α的抑毒作用能被ＴＮＦ－α申抗和ＩＦＮ－β单抗完全去除,被ＩＦＮ－α单抗部分去除ＴＮＦ－α的抗病毒效应。ＴＮＦ－－α中和试验的结果提示：ＴＮＦ抗病毒活性仍为ＩＦＮ－β诱生所介导。