dam methylation and the initiation of DNA replication on oriC plasmids

Summary Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants. here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts. The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant. However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids. We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oric region.     

Changes in plant growth processes under microgravity conditions simulated by a three-dimensional clinostat

We developed a three-dimensional (3-D) clinostat to simulate a microgravity environment and studied the changes in plant growth processes under this condition. The rate of germination of cress (Lepidium sativum), maize (Zea mays), rice (Oryza sativa), pea (Pisum sativum), or azuki bean (Vigna angularis) was not affected on the clinostat. The clinostat rotation did not influence the growth rate of their roots or shoots, except for a slight promotion of growth in azuki roots and epicotyls. On the contrary, the direction of growth of plant organs clearly changed on the 3-D clinostat. On the surface of the earth, roots grow downward while shoots upward in parallel to the gravity vector. On the 3-D clinostat, roots of cress elongated along the direction of the tip of root primordia after having changed the direction continuously. Rice roots also grew parallel to the direction of the tip of root primordia. On the other hand, roots of maize, pea, and azuki bean grew in a random fashion. The direction of growth of shoots was more controlled even on the 3-D clinostat. In a front view of embryos, shoots grew mostly along the direction of the tip of primordia. In a side view, rice coleoptiles showed an adaxial (toward the caryopsis) while coleoptiles of maize and epicotyls of pea and azuki bean an abaxial curvature. The curvature of shoots became larger with their growth. Such an autotropism may have an important role in regulation of life cycle of higher plants under a microgravity environment.     

Primordial germ cells and lymphomyeloid system in the embryos of the Aleutian skate,Bathyraja aleutica

Organogenesis in embryos (1 and 5 mm-disk widths) of the Aleutian skate,Bathyraja aleutica was described histologicaily in complete serial paraffin sections. Special attention was paid to localization of primordial germ cells (PGC) and development of lymphoid tissues. The embryo of 1 mm-disk width was at the somite stage with no evidence of organogenesis. PGC, gathered in the genital ridge, were seen in the somatic mesodermal layer as well as in the mesenchyme between the endoderm and splanchnic mesoderm of the 1 mm-embryo. Most of the visceral organs were developing in the embryo of 5 mm-disk width. With respect to the development of immune system, two pairs of thymus anlagen, filled with numerous thymic lymphocytes, were recognized in the pharyngeal region. A small focus of numerous immature blood cells appeared to be an anlage of the spleen was found between the stomach and the liver.     

Recognition sites for a membrane-derived DNA binding protein preparation in the E. coli replication origin

Summary The DNA binding protein B' preparation, isolated from the membrane of E. coli, recognizes two sites, one of which is locatd in the minimum oriC (35–270 bp) and the other between base pairs 417 and 488. Recognition is only possible when restriction fragments containing these sites are in single-stranded state. At the first site the strand reading 3OH-5P in the direction of the E. coli genetic map is recognized, at the second site the 5P-3OH strand.     

Development of the echinate pollen wall inFarfugium japonicum (Compositae: Senecioneae)

Development of the echinate pollen grains inFarfugium (Compositae: Senecioneae) has been studied by a combination of transmission electron microscopy and field emission scanning electron microscopy with a freeze fractured method. The inner surface of the callose wall surrounding each microspore does not possess an echinate pattern before primexine deposition begins. The primexine formation coincides with the initiation of spines. The freeze fractured primexine shows probacula which form transverse rods. The developing exine has an inner spongy substructure. The endexine is formed by the accumulation of the electron dense lamellae with white lines after the dissolution of the callose wall. In the present study, it is confirmed that the developmental process of pollen formation revealed in the field emission scanning electron microscope is consistent with the results obtained using the transmission electron microscope.     

A lea-class gene of tomato confers salt and freezing tolerance when expressed in Saccharomyces cerevisiae

During periods of water deficit, plants accumulate late embryogenesis-abundant (LEA) proteins which are thought to protect cells from stresses associated with dehydration. One of these genes, le25, is expressed in tomato leaves and roots in response to water deficit and abscisic acid accumulation. To study the function of this protein and to test the effect of overproduction of the LE25 protein in Saccharomyces cerevisiae (Sc), a recombinant plasmid in which le25 is expressed under the control of the GAL1 promoter was constructed. The content of LE25 was high in Sc cells transformed with the recombinant plasmid. The transformant exhibited several stress-tolerant phenotypes. Growth of the transformant in a medium with 1.2 M NaCl was improved, as compared to a control strain. While the control strain showed a long lag phase of 40 h, le25-expressing cells showed a shortened lag phase of 10 h. However, no growth improvement was observed in a medium with 2 M sorbitol. In addition, the transformant had an increased survival rate after freezing stress, but not after high-temperature stress. These results, together with its predicted secondary structure, may indicate that LE25 functions as an ion scavenger.     

Engineering of closed ecological system in space and inter-organismal interactions.

Space agriculture is a concept of synthesis and operation of closed ecological system for controlling living environment and supplying materials in order to sustain life and to meet metabolic needs of space crew. It revitalizes metabolites and other excretion of crew for their recycled usage. It is an advanced concept for life support for long and large scaled manned space missions, where open loop system for materials cycle is not feasible to apply. Several issues, such as cost-benefit analysis with considering benefits of amenity and psychological factors of crew, are discussed in this essay, together with reliability and productivity of biological systems. Studies on plant physiology for inter-organismal interaction contribute to design work for space agriculture, and associate with our engagements to our future, sustainable development of our civilization both on the earth and extending to outer space.