一种简便、高效、经济的从凝胶中回收DNA的方法

目的:尝试一种简便、高效的从凝胶中回收DNA的方法。方法:在Eppendorf管的管底用注射器扎孔,将一小团用Eppendorf管融化后拉成的细丝放入管中,把含有DNA的凝胶放在细丝上,离心,收集从管底流出的液体,经酚氯仿抽提后用乙醇沉淀DNA。结果:经过简单的离心即可近乎100%地回收凝胶中的DNA。结论:使用该方法从琼脂糖凝胶回收DNA,操作简单,回收率高,无其他试剂污染。     

Unexpected distribution of immobilized microorganisms within alginate beads

Immobilization refers to the prevention of free cell movement by natural or artificial means. It has always been assumed that immediately after an immobilization procedure is performed, cells are distributed homogeneously in the beads that entrap them. However, in this study, Escherichia coli and Trichoderma asperellum distribution in alginate-gel beads was found to be nonhomogeneous. In fact, there was a greater presence of cells on the surface of the alginate beads than in their cores.     

A synergistic reaction mechanism of a cycloalternan-forming enzyme and a D-glucosyltransferase for the production of cycloalternan in Bacillus sp. NRRL B-21195

Cycloalternan-forming enzyme (CAFE) was first described as the enzyme that produced cycloalternan from alternan. In this study, we found that a partially purified preparation of CAFE containing two proteins catalyzed the synthesis of cycloalternan from maltooligosaccharides, whereas the purified CAFE alone was unable to do so. In addition to the 117 kDa CAFE itself, the mixture also contained a 140 kDa protein. The latter was found to be a disproportionating enzyme (DE) that catalyzes transfer of a D-glucopyranosyl residue from the non-reducing end of one maltooligosaccharide to the non-reducing end of another, forming an isomaltosyl residue at the non-reducing end. CAFE then transfers the isomaltosyl residue to the non-reducing end of another isomaltosyl maltooligosaccharide, to form an alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-(1-->4)-maltooligosaccharide, and subsequently catalyzes a cyclization to produce cycloalternan. Thus, DE and CAFE act synergistically to produce cycloalternan directly from maltodextrin or starch.     

Comparison of plastid DNA replication in different cells and tissues of the rice plant

In a previous study, we mapped replication origin regions of the plastid DNA around the 3 end of the 23S rRNA gene in rice suspension-cultured cells. Here, we examined initiation of the plastid DNA replication in different rice cells by two-dimensional agarose gel electrophoresis. We show for the first time, to our knowledge, that the replication origin region of the plastid DNA differs among cultured cells, coleoptiles and mature leaves. In addition, digestion of the replication intermediates from the rice cultured cells with mung bean nuclease, a single-strand-specific nuclease, revealed that both two single strands of the double-stranded parental DNA were simultaneously replicated in the origin region. This was further confirmed by two-dimensional agarose gel analysis with single-stranded RNA probes. Thus, the mode of plastid DNA replication presented here differs from the unidirectional replication started by forming displacement loops (D-loops), in which the two D-loops on the opposite strands expand toward each other and only one parental strand serves as a template.     

琼脂糖凝胶直接杂交快速鉴定低拷贝数转基因

采用常规的PCR方法检测低拷贝数转基因有一定困难.提出一种使用琼脂糖凝胶直接杂交的方法,结果明确、简单可行,是转基因动物中低拷贝数转基因鉴定的一种较好方法.     

Effects of bacterial agarase on agarose gel in cell culture

Summary Bacterial agarase, concentrated and purified from culture filtrate of agar-degrading bacteria, has been used to clean cells cultured in soft agarose from gel residues. The enzyme also has been used to liquefy the gel directly in the dishes to facilitate the removal of cells. The surfaces of glioma cells from agarase-treated colonies could not be distinguished in the scanning electron microscope from surfaces of cells which had never been in contact with agarose or agarase. This implies that most agarose residues had been removed, and also that the treatment did not seriously alter the cell surfaces. The influence of the agarase treatment also was tested by comparison of the mitotic index and the incorporation of [³H]thymidine in agarase-treated and untreated cells. No effects of the treatment could be seen in these tests. The work has been supported by the Swedish Cancer Society and the Swedish Natural Science Research Council.     

The use of agarose in the determination of anchorage-independent growth

Summary At the present time, growth in agar suspension is one of the best in vitro correlates of tumorigenicity. Growth in agarose, however, has not been evaluated extensively as an in vitro criterion for tumorigenicity. In the present study we have tested 19 cell lines, including six mouse-human hybrids, for growth in agarose and agar in the presence and absence of exogenous hypoxanthine. None of the six nontumorigenic cell lines grew in agar or agarose. Ten of the 13 tumorigenic cell lines grew in both agar and agarose with about equal efficiency. The remaining three tumorigenic cell lines grew well in agarose but poorly or not at all in agar. Hypoxanthine did not stimulate the growth in agar or agarose of any of the cell lines except BHK. We conclude that growth in agarose may be a more sensitive marker for tumorigenicity than growth in agar and that BHK is exceptional in its sensitivity to supplemental purines. This research was supported by DHEW NCI Contract No. NO-CP-2-3234 and by USPHS Medical Scientist Training Grant GM 02042-07.     

Replication Fork Barriers and Topological Barriers: Progression of DNA Replication Relies on DNA Topology Ahead of Forks

During replication, the topology of DNA changes continuously in response to well-known activities of DNA helicases, polymerases, and topoisomerases. However, replisomes do not always progress at a constant speed and can slow-down and even stall at precise sites. The way these changes in the rate of replisome progression affect DNA topology is not yet well understood. The interplay of DNA topology and replication in several cases where progression of replication forks reacts differently to changes in DNA topology ahead is discussed here. It is proposed, there are at least two types of replication fork barriers: those that behave also as topological barriers and those that do not. Two-Dimensional (2D) agarose gel electrophoresis is the method of choice to distinguish between these two different types of replication fork barriers.