Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism.     

Circular Arrangement of Cortical F-actin around the Subapical Region of a Tip-Growing Fern Protonemal Cell

F-actin organization in the tip-growing cells of fern protonematawas investigated by rhodamine-phalloidin staining in two species:Adiantum capillus-veneris and Pteris vittata. Circular arrangementof cortical F-actin was found around the subapical region ofprotonemal cells in both species. In rhizoids, such structureswere absent and the axial filaments appeared to fan out fromthe tip. (Received May 22, 1989; Accepted September 6, 1989)     

Chloroplast proliferation based on their distribution and arrangement inAdiantum protonemata growing under red light

Chloroplast proliferation was investigated inAdiantum protonemata growing under continuous red light. Cell division is absent when cells are grown under red light. The chloroplast number increases as the cell length increases, therefore the chloroplasts divide in the absence of cell division. Chloroplasts in the basal part of the filamentous protonemal cell migrate gradually toward the cell apex, but there is no large net migration from the tip to the base or vice versa, indicating that chloroplast division takes place in the apical part of the protonemata. Chloroplast number in the apical 100 μm was maintained at about 200 during cell growth at least over eight days. The chloroplasts were either dumbbell- or ellipsoid-shaped. Dumbbell-shaped chloroplasts are abundant everywhere in a protonema, ranging from 30 to 50% of the total chloroplasts. The dumbbell-shaped chloroplasts attached to or very close to the plasma membrane seem to be the ones that are dividing but the dumbbell-shaped ones in the other regions do not divide. These data support the hypothesis that a signal from the plasma membrane induces the dumbbell-shaped chloroplasts to divide.     

Sensitive detection of viral antigens with a new method, "laser magnet immunoassay".

A new method, "laser magnet immunoassay" (LMIA), has been developed for sensitive detection of viral antigens. Target viruses captured on microbeads were made to react with antibodies labeled with magnetite particles. In a magnetic field, magnetically labeled antigens dispersed in water were attracted to and concentrated at one point on the surface, resulting in the lifting up of a small surface area. A laser beam which was incident on the point reflected, making an interference fringe. The intensity of the fringe indicates the amount of the magnetite conjugated with antigen. A very low concentration of antigens, such as 5 particles of influenza virus and 0.1 pg/ml of human immunodeficiency virus (HIV) p24 antigen in human serum, could be detected by this method. Application of this method to diagnoses of viral diseases in early stages is discussed.     

Occurrence of lymphohaemopoietic tissue in the meninges of the stingray Dasyatis akajei (Elasmobranchii, chondricthyes)

The cytoarchitecture of the lymphohaemopoietic masses occurring in the "meninx primitiva" of the stingray Dasyatis akajei (Elasmobranchii, Chondricthyes) has been analyzed by light and scanning and transmission electron microscopy. Lymphohaemopoietic aggregates showing similar morphologies occurred along all the central nervous system, but they were more frequent in the telencephalon, diencephalon, and mesencephalon. In each aggregate, the granulopoietic tissue appeared in a fibroblastic stroma surrounding the large blood vessels, and the lymphoid components were present in a reticular network. Developing and mature eosinophils and heterophils--as well as lymphocytes, monocytes, macrophages, and plasma cells--are the main free cells present in these meningeal aggregates. The remarkable intimate association between macrophages and lymphoid cells to form close cell clusters suggests some immunological capacity for the meningeal lymphohaemopoietic tissue. According to their capacities, presence of lymphoid tissue, and histological organization, the meningeal lymphohemopoietic aggregates of Dasyatis akajei resemble other lymphomyeloid aggregates associated with cranium and choroid plexuses in Holocephali and Ganoidei. The phylogenetical relationships of these aggregates with mammalian bone marrow are discussed.     

Immunohistochemical localization of plasma retinolbinding protein and prealbumin in human pancreatic islets

Summary This study was undertaken, employing the immunoenzyme method, to confirm the presence of retinol-binding protein in human pancreatic islets, and to compare its distribution with that of prealbumin, insulin, glucagon, somatostatin and pancreatic polypeptide. It was found that most islet cells contained retinol-binding protein, although centrally located cells showed stronger reactivity than those in the peripheral region. The distribution of each of the five polypeptides differed from that of retinolbinding protein, indicating that these peptides did not cross-react with anti-retinol-binding protein antibody. Islet cells which contained prealbumin, on the other hand, were mostly classified as A cells. Further studies are necessary to confirm whether the islet cells produce retinol-binding protein or only store it.     

NAD+ Glycohydrolase of the Plasma Membrane Prepared from Glial and Neuronal Cells

NAD+ glycohydrolase (EC 3.2.2.5) activity was detected in the plasma membrane prepared from the primary culture of rat astrocytes. The enzyme has a broad optimum pH range. From the kinetic analysis, a Michaelis constant of 91.2 microM and a maximum velocity of 0.785 mumol/min/mg protein were obtained. ADPribose exhibited a competitive inhibition with respect to NAD. The inhibition by nicotinamide was shown to be of a non-competitive type. ATP and GTP were found to be competitive inhibitors. NAD+ glycohydrolase activity was not detected in the plasma membrane prepared from the primary culture of neuronal cells of chick embryos.     

Blue Light-Induced Phototropic Response and the Intracellular Photoreceptive Site in Adiantum Protonemata

Blue light-induced phototropism in Adiantum protonemata wasinvestigated with microbeam irradiation. Brief irradiation withblue light effectively induced a phototropic response when itwas applied to a half-side of the apical 200d µm regionof a protonema. The phototropic response was partly reversedby the subsequent far-red light irradiation but the full reversalof the response was not observed even when the fluence of far-redlight was increased. In the far-red reversible part of the response,blue/far-red photoreversibility was repeatedly observed. Thus,both phytochrome and a blue light-absorbing pigment (other thanphytochrome) seem to be involved in the blue light-induced phototropicresponse in Adiantum protonemata. Furthermore, detailed studiesof the far-red light effect on the fluence-response curve forblue lightinduced phototropism revealed that the concomitantmediation by the two receptors was limited to the response inthe relatively higher fluence range of blue light and that theblue light-absorbing pigment alone was responsible in the lowerfluence range. In the higher fluence range, the response mediatedby the blue light-absorbing pigment became saturated and thephytochrome response became evident, indicating a differencein the sensitivities of the two receptor pigments to blue light. When various regions of half-sides of protonemata were irradiatedwith a blue microbeam of 10 µm width, irradiation at theapical 5–25 µm region was most effective both forphytochrome- and blue light-absorbing pigment-mediated response,indicating that the site of blue light perception is almostidentical for each response. (Received July 14, 1986; Accepted September 26, 1986)