核酸去甲基酶AlkB在毕赤酵母中的表达纯化及在tRNA相关研究中的应用

[目的]核酸的甲基化修饰是一种常见的化学修饰形式,具有重要的生物学功能,却也在一定程度上给一些核酸研究过程带来了技术难度。tRNA上具有的大量甲基化修饰会阻碍逆转录进程,从而降低荧光定量PCR (real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)和高通量测序对其的检测效率。来自大肠杆菌(Escherichia coli)的AlkB蛋白是一种多功能的脱烷基化酶,可以去除DNA和RNA上多种甲基化为代表的修饰,有望解决以上问题。[方法]针对大肠杆菌来源的AlkB,分别尝试在大肠杆菌和毕赤酵母(Pichia pastoris)表达系统中进行诱导表达和纯化,对纯化获得的AlkB进行酶学性质测定。最后以tRNA_UAU^Ile等两种tRNA为代表,研究AlkB的处理对于荧光定量PCR法检测tRNA表达水平的影响。[结果]AlkB在大肠杆菌中表达时多以包涵体形式存在,但是在毕赤酵母中可以成功分泌表达。使用镍柱分离纯化后获得了纯度高于95%的AlkB蛋白,其酶学性质参数如...     

The mosquito dihydrofolate reductase gene functions as a dominant selectable marker in transfected cells

An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors that functioned as dominant selectable markers in transfected, wild type mosquito cells. Stably transformed clones were recovered after 10–15 days in the presence of selective medium containing 1 μM methotrexate. The transformed clones contained an estimated 100–500 copies of transfected DNA per nucleus. Combined data from Southern blots and in situ hybridization to metaphase chromosomes indicated that transfected DNA was likely integrated into chromosomes both as repeated structures and as randomly integrated single copy molecules, with minimal rearrangement of coding sequences. Transfected DNA was stably maintained under selective conditions, but in some cases was lost when cells were maintained for prolonged periods in the absence of methotrexate. These observations provide a general framework for further development of stable gene transfer systems for mosquito cells in culture.     

Epithelial M cells in the rabbit caecal lymphoid patch display distinctive surface characteristics

The follicle-associated epithelium (FAE) in the rabbit caecal lymphoid patch is characterised by the presence of membranous (M) cells, which are believed to be functionally equivalent to those present at other sites of gut-associated lymphoid tissue (GALT). Caecal patch M cells display distinctive features compared with those of other GALT sites, despite similar general morphology and expression of the M cell marker vimentin, suggesting marked heterogeneity in the apical surface of M cells at discrete GALT sites. Electron microscopy reveals that rabbit caecal patch M cells differ from those in the small intestinal Peyer's patch FAE: the former have a prominent aspect within the epithelium and possess microvilli which are longer than those of adjacent enterocytes. Many of the M cells in peripheral regions of the caecal patch FAE are not associated with leucocytes and may thus represent an immature M cell population. The M cells are also histochemically distinct from adjacent enterocytes and from Peyer's patch M cells, showing greater expression of brush-border alkaline phosphatase activity and affinity for certain lectins (peanut and wheat germ agglutinins, Bandeiraea simplicifolia agglutinin II). The differences in the brush-border morphology and glycocalyx structure between M cells at different GALT sites may affect their function at these sites by influencing the interaction of luminal antigens and microorganisms with the M cell surface. The present data also support the hypothesis that M cells arise directly from differentiation of crypt stem cells and not from the transformation of existing fully differentiated enterocytes.     

The Thymic Microenvironment of the Common Sole,Solea solea

Abstract The thymus of the sole Solea solea contained lymphoblasts and thymocytes within a network of pale and dark epithelial cells. The pale cells were characterized by tonofilaments and desmosomes and some embraced rodlet cells within their cytoplasmic processes. The dark epithelial cells had numerous electron-dense inclusions and electron-lucent vacuoles. Lymphocytes were closely associated with the plasma membrane of both types of epithelial cells and with macrophages. Breakdown of effete lymphocytes appeared to be the main function of the macrophages. Some macrophages were multinucleated. Those containing melanin granules associated with phagosomes were classified as melanomacrophages. Pigment cells including melanophores and guanophores were present along the connective tissue trabeculae and surrounding the blood vessels. A few plasma cells and mucous cells were present.     

ECOLOGICAL ASPECTS OF SEX EXPRESSION IN SUBDIOECIOUS SCHIEDEA GLOBOSA (CARYOPHYLLACEAE)

The importance of ecological factors such as sex lability, spatial segregation, and resource allocation in the evolution of dioecy were examined in Schiedea globosa. S. globosa is a subdioecious species with equal numbers of plants possessing strictly male or female function and a small proportion of hermaphrodites. The propensity for labile sex expression was under both environmental and genetic control; some plants with male function became hermaphroditic (by producing female flowers) under better growing conditions in the field and in the greenhouse. There was some spatial segregation of the sexes. Because of sex lability, more hermaphrodites than males occurred on moister slopes. Although there were not measurable sex-related differences in mortality within or between two flowering seasons, more females than males and hermaphrodites occurred at the bottom of slopes. Males and females produced the same number of ramets and inflorescences, but females had a greater number of flowers per inflorescence. Males and females had the same number of ovules (vestigial in males), but females had larger ovules and longer stigmas. Hermaphrodites and males had the same amount of pollen per flower despite the production of fruit by the hermaphrodites. In hermaphrodites, there was no apparent tradeoff within flowers between pollen production and ovule production. These results indicate that spatial segregation, sex lability, and environmental conditions influence allocation patterns of S. globosa, and in combination with high inbreeding depression and selling rates, may promote the further evolution of dioecy in S. globosa.     

REPRODUCTIVE SEASONALITY OF WESTERN ATLANTIC BOTTLENOSE DOLPHINS OFF NORTH CAROLINA, U.S.A.

We describe reproductive seasonality of bottlenose dolphins in North Carolina (NC), U.S.A., using strandings data from the entire coast of NC and sighting data from Beaufort, NC and by estimating dates of birth of known females. We found a strong peak of neonate strandings in the spring (April-May), and low levels of neonate strandings in the fall and winter. The distribution of neonate strandings was significantly different from a uniform distribution ( P < 0.001, K = 3.8). We found a unimodal distribution of 282 sightings of neonates with a diffuse peak in the summer. The temporal distribution of sightings of neonates departed significantly from a uniform distribution (P < 0.001, K = 5.1). Estimated birth dates of neonates from known females occurred in May ( n = 6) and June ( n = 4), with a single fall birth. These methods shed light on bottlenose reproductive patterns and underscore the value of using information from multiple types of data. Clarification of bottlenose dolphin reproductive patterns, such as the seasonality of birth, may enhance our understanding of the population structure of this species in the mid-Atlantic region.     

Expression of wild-type GBSS transgenes in the off-spring of partially and fully complementedamylose-free transformants of potato

Theamylose-free (amf) potato mutant can easily be complemented through introduction of the wild-type gene coding for granule-bound starch synthase (GBSS). After iodine staining the starch of theamf mutant is red whereas that of the wild type and the complementedamf mutant is blue. The level of complementation of selected transformants and their sexual off-spring after backcrossing withamf was investigated using sporophytic tuber cells and gametophytic microspore cells. Two diploid and two tetraploid transformants with full complementation demonstrated the expected segregation patterns of 1:1 (one active insert) or 3:1 (two independently segregating active inserts) in the microspores and in the F1 offspring based on staining of tubers. All expected genotypes in the F1 generation were found, based on microspore segregation patterns of the individual F1 plants. Two transformants with partial complementation (mixed phenotypes) were investigated. One of them, B1, was tetraploid and duplex for the GBSS insert, which had originated through mitotic doubling of the transformed diploid cells. In the F1 generation three phenotypic classes were found:amf, fully complemented and partially complemented. The latter two classes exist independently of a simplex or duplex gene status. The second transformant with partial complementation, B10, appeared to have a complex molecular composition. One cluster of five transgenes caused the partial complementation. Fully and partially complemented phenotypic classes were found after crossing B10 with theamf mutant. Indications were found that the ploidy level of the tissue in which the genes were introduced and expressed played an important role. Firstly, partial complementation was found after transformation of the diploid and not of the tetraploidamf genotypes. Secondly, the level of complementation was higher in tissue with lower ploidy levels, as illustrated by the colour of the starch inin vitro tubers (2x–4x cells) versus field-grown tubers (16x–64x).