Identification of essential residues in Thermus thermophilus DNA ligase.

DNA ligases play a pivotal role in DNA replication, repair and recombination. Reactions catalyzed by DNA ligases consist of three steps: adenylation of the ligase in the presence of ATP or NAD+, transferring the adenylate moiety to the 5'-phosphate of the nicked DNA substrate (deadenylation) and sealing the nick through the formation of a phosphodiester bond. Thermus thermophilus HB8 DNA ligase (Tth DNA ligase) differs from mesophilic ATP-dependent DNA ligases in three ways: (i) it is NAD+ dependent; (ii) its optimal temperature is 65 instead of 37 degrees C; (iii) it has higher fidelity than T4 DNA ligase. In order to understand the structural basis underlying the reaction mechanism of Tth DNA ligase, we performed site-directed mutagenesis studies on nine selected amino acid residues that are highly conserved in bacterial DNA ligases. Examination of these site-specific mutants revealed that: residue K118 plays an essential role in the adenylation step; residue D120 may facilitate the deadenylation step; residues G339 and C433 may be involved in formation of the phosphodiester bond. This evidence indicates that a previously identified KXDG motif for adenylation of eukaryotic DNA ligases [Tomkinson, A.E., Totty, N.F., Ginsburg, M. and Lindahl, T. (1991) Proc. Natl. Acad. Sci. USA, 88, 400-404] is also the adenylation site for NAD+-dependent bacterial DNA ligases. In a companion paper, we demonstrate that mutations at a different Lys residue, K294, may modulate the fidelity of Tth DNA ligase.     

生物复苏——大绝灭后生物演化历史的第一幕

生命史是一部生物界短期,快速剧变与长期,慢速稳定相互交替的历史。大绝灭（即集群绝灭）事件反映了全球环境的大突变,点断了地质历史中的生命记录及其发展历程,预示着生物界的演化出现了最有意义的飞跃,近年来尝试研究大绝灭后全球生物界的残存－复苏及其基本型式,并探索复苏的控制因素,标志着地质科学中一个重心的转移（即从大绝灭转向其后的生物残存与复苏的研究）。生物复苏揭示了大绝灭后生物演化历史的第一幕,其研究的     

广西、贵州的Zelia及其地层分布

标本采自广西宜山、天等和贵州独山、紫云等地的早二叠世地层中。Ｚｅｌｌｉａ富集于层面,大多和Ｐｓｅｕｄｏｓｃｈｗａｇｅｒｉｎａ,Ｓｐｈａｅｒｏｓｃｈｗａｇｅｒｉｎａ,Ｒｏｂｕｓｔｏｓｃｈｗａｇｅｒｉｎａ共生,在确定石炭二叠系界线时具有一定的地层意义。现对Ｚｅｌｉａ的属征加以阐述、讨论和归并,并对其地理地层分布作一简介     

丝孢酵母高甲硫氨酸突变株的选育及营养调控

以丝孢酵母(Trichosporon Behr)ST851为原始菌株,经紫外线诱变,在含乙硫氨酸的双层平板上筛选到多株抗乙硫氨酸突变株。其中ST851-10株抗乙硫氨酸浓度达到350μg/ml,其菌体蛋白质含量由40.5％提高到44.3％,菌体甲硫氨酸含量由20.45mg/g-DCW增加到29.32mg/g-DCW。在以苹果渣为碳源、尿素为氮源、硫酸镁作硫源的最适培养条件下,固态发酵24h后,蛋白质和甲硫氨酸含量较原始菌株分别提高了15.8％和44.9％。培养基中C/N值低有利于甲硫氨酸的合成,C/N值高则适合于菌体生长。在苹果渣固态发酵过程中,适当补加氮源既有利于菌体生长和甲硫氨酸的合成,又可起到调节培养基pH值的作用。     

太湖16000年来沉积环境的演变

通过对太湖钻孔岩芯岩性,结构,构造的剖析及粒度,磁化率的测试,发现冰后期东太湖形成于跑今６５００年前,在距今６５００－５８００年,为一水深约２－３ｍ的,经常受到流水作用影响的浅水湖泊,距今约５８００－５７００年,东太湖曾一度干枯或接近于干枯,距今５７００年以来湖泊变浅,平均水深只有１ｍ左右,由于湖泊变浅,湖底经常遭受波浪的扰动,形成波状层理或透镜状层理。西太湖局部洼地集水成湖的时间比东太湖早,并且     

Mitochondrial DNA polymorphism and phylogenetic relationships inHevea brasiliensis

Using fourteen random mitochondrial DNA probes, we have examined restriction fragment length polymorphism (RFLP) in wild and cultivatedHevea brasiliensis. A total of 395 accessions, including 345 from various prospectings collected in Brazil, Colombia and Peru and 50 cultivated clones, were analyzed. Two other species (H. benthamiana andH. pauciflora) were also included in the study for comparison. The high level of mitochondrial polymorphism allowed us to divide all the accessions analyzed into 212 distinct genotypes. The genetic variability of cultivated clones was limited to four genotypes forming two clusters. In contrast, considerable genetic variation was found in the wild collections. In almost all cases, accessions displaying the same RFLP profile were restricted to the same geographical area (same or neighbor administrative districts). In addition, accessions whose genetic closeness was predicted by RFLP profiles were also clustered according to geographical origin. In a few cases, however, similar RFLP profiles were found for accessions originating from geographically distant districts. This discrepancy can be explained either by seed dispersion (by river) or possibly by similar genetic events occurring independently in different geographical locations. Chloroplast DNA RFLP was also analyzed in 217 accessions, representative of 126 distinct mitochondrial genotypes. Very few differences were found, indicating that the chloroplast genome is more highly conserved than the mitochondrial genome.     

猪精子凝集素的纯化,性质及其作用

用胎球蛋白－Ｓｅｐｈａｒｏｓｅ亲和层析和凝胶过滤层析从精子和精浆中分离纯化了猪精子凝集素（简称ＢＳＬ）。ＢＳＬ的血凝活性只被若干糖蛋白和聚糖所抑制。ＢＳＬ的分子量为５６ｋｄ,由分子量分别为１３．６ｋｄ（β）和１６．０ｋｄ（α）的两个不同的亚基以α１β３所组成。ＢＳＬ为糖蛋白,含中性糖３．２％,不含唾液酸。用ＥＬＩＳＡ法测定猪精子中ＢＳＬ的含量及分布,表明７０％嵌入在精子膜中,２５％结合在精子表面,     

利用激光微束穿刺法将外源基因导入小麦的研究

用激光微束穿刺法将携带有新霉素磷酸转移酶（ＮＰＴ－Ⅱ）基因的质粒ｐＪＩＴ１０１导入京花１号小麦幼胚细胞。方法是将小麦幼胚细胞进行高渗缓冲液预处理,用微米级的激光微束处理,然后在卡那霉素培养基上筛选出具抗性的愈伤组织及绿色小植株。第一年,从１５０个小麦幼胚中,在卡那霉素培养基上筛选出４株绿苗,取两株进行ＮＰＴ－Ⅱ酶活性分析,测到了ＮＰＴ－Ⅱ酶的活性。第二年重复实验,从２４５个小麦幼胚中,经筛选获得１株绿苗,进行了叶片ＤＮＡＰＣＲ扩增检测,转化的绿色小苗扩增出所导入的ＮＰＴ－Ⅱ基因编码的片段。结果表明,外源ＮＰＴ－Ⅱ基因已导入了小麦,并实现了整合表达。     

三种鱼类生长激素cDNA基因的结构比较研究

从厦门海区选取３种生长速度不同的鱼类,真鲈、高体Ｓｈｉ、褐菖Ｙｏｕ,由它们的脑下垂体中分别提取出总ＲＮＡ,用逆转录ＰＣＲ方法（ＲＴ－ＰＣＲ）扩增出生长激素ｃＤＮＡ,克隆到ｐＢｌｕｅｓｃｒｉｐｔ载体上的ＥｃｏＲＩ位点,并分别测定了这３种鱼的成熟生长激素ｃＲＮＡ序列。将由这３种序列推导出的氨基酸序列同已知的８种不同科鱼类的生长激素氨基酸序列进行比较,分析它们序列的同源性,结果表明它们间的同源性与这些鱼     

应用PCR技术检测细小病毒H－1DNA在人肝癌与裸鼠正常组织中复制的差异

应用ＰＣＲ技术检测细小病毒Ｈ－１ＤＮＡ在人肝癌与裸鼠正常组织中复制的差异黄青山,马承武,郭兰萍,陈献华,罗祖玉（上海复旦大学生理与生物物理学系,上海２００４３３）关键词：自主性细小病毒Ｈ－１及ＭＶＭ,聚合酶链式反应（ＰＣＲ）,人肝癌模型,抑瘤作用,肿...