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.     

Glutamine Synthetase of the Human Brain: Purification and Characterization

Glutamine synthetase (GS) isolated from human brain formed a single band on sodium dodecyl sulfate-polyacrylamide gel with a molecular weight of 44,000. The enzyme had a specific activity of 179.2 U/mg protein when assayed by measuring the rate of the formation of gamma-glutamylhydroxamate using hydroxylamine as a substrate. In the presence of manganese ions, the relative activity of human brain GS was much lower than that of the sheep brain enzyme. The suppression of activity by increasing the ADP concentration, however, was less marked in the human enzyme than that in the sheep enzyme. Antibodies were raised in rabbits against the purified enzyme. The double-immunodiffusion technique disclosed cross-reactivities among GSs isolated from human, sheep, and rat brains, but the enzymes were not immunologically identical. Immunohistochemically, GS was localized in the cytoplasm of astrocytes in the human and rat brains and in pericentral hepatocytes of the liver.     

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)     

Spontaneous excretion of D-alanine in urine in mutant mice lacking D-amino-acid oxidase.

Urine from mutant mice lacking D-amino-acid oxidase contained a large amount of alanine compared with that from normal mice. Urinary alanine of the mutant mice was sensitive to D-amino-acid oxidase. H.p.l.c. showed that about 94% of the urinary alanine had the D-configuration. These results suggest that D-amino-acid oxidase functions to decompose D-amino acid(s) in normal mice.     

Involvement of D-amino acid oxidase in elimination of free D-amino acids in mice.

The physiological role of D-amino acid oxidase was investigated by using mutant ddY/DAO- mice lacking the enzyme. Free D-amino acid concentrations in the mutant mice were significantly higher than those of control ddY/DAO+ mice in kidney, liver, lung, heart, brain, erythrocytes, serum and urine. The results suggest that the enzyme is involved in the catabolism of free D-amino acids in the body, and that free D-amino acids are also excreted into urine.     

Intestinal bacterial origin of D-alanine in urine of mutant mice lacking D-amino-acid oxidase.

Urine from mutant mice which lack D-amino-acid oxidase contained a large amount of D-alanine. The alanine content was not changed by changes in the dietary composition or by starvation. However, oral administration of an antibiotic, amoxicillin, decreased the urinary alanine to the normal level. These results suggest that the D-alanine is not of dietary origin but of intestinal bacterial origin.     

Intraoperative imprint cytology of the thyroid gland with computer-assisted morphometric analysis of cell clusters

Imprint preparations were used in addition to frozen sections in the intraoperative diagnosis of 37 cases of benign and malignant lesions of the thyroid gland, including adenomatous goiter, follicular adenoma, follicular carcinoma and papillary carcinoma. In the imprints, the cytologic features specific for carcinoma, as compared with benign lesions, were (1) the folding of the nuclear contour, (2) the increased density of the cytoplasmic matrix and (3) the frequent appearance of cell clusters of larger size. The size and frequency of cell clusters were morphometrically analyzed by a computer image analyzer. There was an increasing number of large clusters, plus the appearance of clusters of more than 300 micron in diameter, in both follicular and papillary carcinoma. In benign lesions, on the contrary, the majority of cells were isolated or in small clusters, the diameter of which never exceeded 300 micron in diameter. These results demonstrate that (1) the imprint cytology of the thyroid gland is useful in making a rapid intraoperative diagnosis and (2) the introduction of computer-assisted quantitative analysis is of practical value in the diagnosis of malignancy.     

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.     

A case of Graves' disease associated with an autonomously functioning thyroid nodule (AFTN) (Marine-Lenhalt syndrome) which spontaneously became a cold nodule

We report a 31-year-old female with Graves' disease associated with an autonomously functioning thyroid nodule (AFTN) (Marine-Lenhalt syndrome) in which the AFTN spontaneously became a cold nodule. Initially the patient was thyrotoxic and had diffuse goiter with an elevated radioiodine uptake. She became euthyroid following six months of antithyroid drug therapy, and in addition to diffuse goiter, the solitary hot nodule was palpable in the left lobe. Fourteen months later, hyperthyroidism recurred and the thyroid scan revealed diffuse radioiodine uptake with a cold area in the nodular region. The resected nodule showed extensive degeneration and the histological diagnosis was follicular adenoma with Graves' disease. We discussed the significance of recognizing the syndrome and also compared the frequency of spontaneous degeneration in AFTN and in solitary cold nodules.