Cloning and sequence analysis of the StsI restriction-modification gene: presence of homology to FokI restriction-modification enzymes.

StsI endonuclease (R.StsI), a type IIs restriction endonuclease found in Streptococcus sanguis 54, recognizes the same sequence as FokI but cleaves at different positions. A DNA fragment that carried the genes for R.StsI and StsI methylase (M.StsI) was cloned from the chromosomal DNA of S.sanguis 54, and its nucleotide sequence was analyzed. The endonuclease gene was 1,806 bp long, corresponding to a protein of 602 amino acid residues (M(r) = 68,388), and the methylase gene was 1,959 bp long, corresponding to a protein of 653 amino acid residues (M(r) = 76,064). The assignment of the endonuclease gene was confirmed by analysis of the N-terminal amino acid sequence. Genes for the two proteins were in a tail-to-tail orientation, separated by a 131-nucleotide intercistronic region. The predicted amino acid sequences between the StsI system and the FokI system showed a 49% identity between the methylases and a 30% identity between the endonucleases. The sequence comparison of M.StsI with various methylases showed that the N-terminal half of M.StsI matches M.NIaIII, and the C-terminal half matches adenine methylases that recognize GATC and GATATC.     

Induction of interdigitating reticulum cell-like differentiation in human monocytic leukemia cells by conditioned medium from IL-2-stimulated helper T-cells

Monocytic leukemia (MoL) cells were obtained from the peripheral blood of a patient in whom the leukemic cells infiltrating various lymphoreticular organs exhibited features intermediate between interdigitating reticulum cells (IDC) and ordinary phagocytic macrophages, whereas the leukemic cells in the peripheral blood were essentially monocytic and lacked such features. Peripheral blood CD4+ T-cells were established as an interleukin-2-dependent T-cell line. When the MoL cells were exposed for a few days to conditioned medium from the T-cell line, they extended several dendritic cytoplasmic projections and became intensely positive for HLA-DR antigen, cytoplasmic S-100β protein, and CD1 antigen. Functionally, the conditioned medium significantly down-regulated Fc-mediated and Fc-independent phagocytic activities, and the levels of lysosomal enzymes such as lysozyme and nonspecific esterase in the MoL cells. Moreover, the conditioned medium significantly up-regulated the accessory cell function of the MoL cells as measured by the primary allogenic mixed leukocyte reaction (MLR). Furthermore, the conditioned medium significantly down-regulated the expression of CD14 antigen. Biochemical analysis indicated that the factor responsible for these changes is a protein which is distinct from known human cytokines and whose molecular weight is approximately 31 kDa. These findings suggest that IDC are closely related the monocytic lineage and that helper T-cells play an important role in constructing the microenvironment of T-lymphoid tissues which is necessary for the differentiation and maturation of IDC.     

Cholinergic differentiation factor (CDF/LIF) promotes survival of isolated rat embryonic motoneurons in vitro.

We present evidence that the cholinergic differentiation factor (CDF), originally purified from cardiac and skeletal muscle cell-conditioned medium and found to be identical to leukemia inhibitory factor (LIF), promotes survival of embryonic day 14 rat motoneurons in vitro. These neurons were retrogradely labeled with the fluorescent tracer Dil and enriched on a density gradient or purified to homogeneity by fluorescence-activated cell sorting. Subnanomolar concentrations of CDF/LIF supported the survival of 85% of the motoneurons that would have died between days 1 and 4 of culture. The enhanced survival was accompanied by a 4-fold increase in choline acetyltransferase (ChAT) activity per culture. CDF/LIF also increased ChAT activity in dorsal spinal cord cultures, but had no detectable effect on ChAT levels in septal or striatal neuronal cultures. For comparison, other neurotrophic molecules were tested on motoneuron cultures. Ciliary neurotrophic factor had effects on motoneuron survival similar to those of CDF/LIF, whereas basic fibroblast growth factor was somewhat less effective. Nerve growth factor had no effect on the survival of rat motoneurons.     

Enclosure experiment on the control mechanism of planktonic bacterial standing stock

In order to understand the control mechanisms of a large, stable bacterial standing stock, enclosure experiments were conducted in a eutrophic lake, where both bacterial productivity and grazing pressure were very high. Total bacterial number in the different enclosures ranged from 1.2 to 2.7×10⁷ cells mL⁻¹ throughout the experiment. The average bacterial cell production rate estimated from a grazer eliminating experiment was 6.3×10⁵ cells mL⁻¹ h⁻¹. Difference in the bacterial cell production rate between shaded and unshaded enclosures was not apparent. Bacteria showed a reduction in standing stock of only about 25–30% even after the supply of light was cut to 1%. Bacteria in the shaded enclosures then recovered their production rate in the first 12 days of perturbation. Grazing pressure in the shaded enclosures was not less than that for the control. Thus, it was considered a control mechanism of bacterial stable standing stock that the bacteria shifted their organic substrate from extracellular dissolved organic carbon freshly released from phytoplankton to that already stocked in the water column, though it is not known whether the dominant bacteria were the same.     

Purification and properties of mycodextranase from Streptomyces sp. J-13-3.

An enzyme hydrolyzing nigeran (alternating alpha-1,3- and alpha-1,4-linked glucan) was purified from the culture filtrate of Streptomyces sp. J-13-3, which lysed the cell wall of Aspergillus niger, by percipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50, CM-Sephadex C-50, chromatofocusing, and Sephadex G-100. The final preparation was homogenous in polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme was 68,000 by SDS-PAGE and gel filtration. The optimum pH and temperature for the enzyme activity were 6.0 and 50 degrees C, respectively. The enzyme was stable in the pH range from 6.0 to 8.0 and up to 50 degrees C. The enzyme activity was inhibited significantly by Hg+, Hg2+, and p-chloromercuribenzoic acid. The Km (mg/ml) for nigeran was 3.33. The enzyme specifically hydrolyzed nigeran into nigerose and nigeran tetrasaccharide by an endo-type of action, indicating it to be a mycodextranase (EC 3.2.1.61) that splits only the alpha-1,4-glucosidic linkages in nigeran.     

Purification of a new extracellular 90-kDa serine proteinase with isoelectric point of 3.9 from Bacillus subtilis (natto) and elucidation of its distinct mode of action.

A new extracellular 90-kDa serine proteinase with an isoelectric point (pI) of 3.9 was purified from Bicillus subtilis (natto). Microheterogeneity was detected in the 50-kDa protease of bacillopeptidase F with pI 4.4 reported previously by Wu et al. and the sequence for the first 25 amino acids in the internal region of the enzyme was analyzed: ATDGVEWNVDQIDAPKAWALGYDGA. The cleavage sites in the oxidized B-chain of insulin by the proteinase were CySO3H7-Gly8, Val12-Glu13, Try16-Leu17, and Phe25-Tyr26. The activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, while the activity was not inhibited by proteinaceous Streptomyces subtilisin inhibitor (SSI) or alpha 2-macroglubulin.     

Phylogeny of gymnosperms inferred fromrbcL gene sequences

Partial nucleotide sequences of the large subunit of ribulose-1,5-bisphosphate carboxylase (rubisco) gene (1333 base pairs: about 90% of the gene) from several seed plants were determined. Phylogenetic trees based on amino acid sequences were inferred by using the neighbor joining and maximum likelihood methods. The results indicate (1) monophyly of gnetum group (Ephedra, Gnetum, Welwitschia), (2) monophyly of extant gymnosperms containing gnetum group, which contradicts the results of morphological data.     

Comparative leaf development ofOsmunda lancea andO. japonica (osmundaceae): Heterochronic origin of rheophytic stenophylly

Comparative development of the narrow pinnules of rheophyticOsmunda lancea and of the broad pinnules of a related dryland species,O. japonica, was examined and the origin of rheophytic stenophylly was discussed. The mature leaves and their various parts ofO. lancea are smaller and narrower than those ofO. japonica. The young pinnules ofO. lancea at the initiation of cell expansion are smaller than those ofO. japonica. The growth pattern of the pinnules is fundamentally the same in the two species, but pinnule growth period is shorter inO. lancea than inO. japonica. While the largest growth rate in pinnule length is quite similar, inO. lancea the pinnules are less elongated and much less broadened during ontogeny. Cell expansion in the mesophyll and epidermis proceeds acropetally and toward the margin along the axes of costules and veins. Although the numbers of mesophyll and epidermal cells between two adjacent veinlets are almost the same inO. lancea andO. japonica, during the subsequent growth period inO. lancea, the cells expand to a smaller extent and the veinlets become more narrowly oblique to the costule. This oblique distortion of laminar segments framed by veins causes stenophylly, an allometric modification. The stenophylly ofO. lancea is believed to have arisen by heterochronic evolution, in particular, progenesis.     

Diploid and triploid offspring of triploid agamosporous fernDryopteris pacifica

A cytological and reproductive study of the diploid and triploid agamosporousDryopteris pacifica was made to elucidate the origin of its infraspecific cytotypes. Some triploids produced 16 spore mother cells (SMCs) sometimes with n=41II+41I chromosomes, in addition to eight SMCs with n=123II, in each sporangium. In the former case the 16 SMCs usually underwent abnormal meiosis to give rise to some 50 spores, some of which were regular-shaped; in the latter the eight SMCs multiplied into 32 spores by normal meiosis. We found that spores from one of the triploid plants developed into either diploid or triploid gametophytes, which further apogamously produced diploid or triploid sporophytes, respectively. This novel mechanism of ploidy reduction is discussed in relation to the origin of diploid agamosporous ferns, the taxonomic complexity of the species, and the correlation of agamospory with polyploidy. The mechanism is also compared to that operating in agamospermous angiosperms.