Hisao Fujisawa

Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant.

Mutations in CUC1 and CUC2 (for CUP-SHAPED COTYLEDON), which are newly identified genes of Arabidopsis, caused defects in the separation of cotyledons (embryonic organs), sepals, and stamens (floral organs) as well as in the formation of shoot apical meristems. These defects were most apparent in the double mutant. Phenotypes of the mutants suggest a common mechanism for separating adjacent organs within the same whorl in both embryos and flowers. We cloned the CUC2 gene and found that the encoded protein was homologous to the petunia NO APICAL MERISTEM (NAM) protein, which is thought to act in the development of embryos and flowers.

Phage DNA packaging

Phage DNA packaging occurs by DNA translocation into a preformed protein shell—a prohead—with the aid of a packaging enzyme or a terminase. The packaging enzyme is composed of two subunits: the large subunit has ATP‐binding, prohead binding, and DNA cleavage activities, and the small subunit is a DNA binding protein. DNA translocation is driven by ATP hydrolysis. In general, phage DNA replication mechanisms lead to the accumulation of concatemers. Concatemers are processed to mature DNA during and depending upon DNA packaging. This review will focus on the molecular mechanism of concatemer processing and the coupling of ATP hydrolysis to DNA translocation.

Gravitropic response of inflorescence stems in Arabidopsis thaliana.

We have characterized the gravitropic response of inflorescence stems in Arabidopsis thaliana. When the inflorescence stems were placed horizontally, they curved upward about 90[deg] within 90 min in darkness at 23[deg]C, exhibiting strong negative gravitropism. Decapitated stem segments (without all flowers, flower buds, and apical apices) also showed gravitropic responses when they included the elongation zone. This result indicates that the minimum elements needed for the gravitropic response exist in the decapitated inflorescence stem segments. At least the 3-min gravistimulation time was sufficient to induce the initial curvature at 23[deg]C after a lag time of about 30 min. In the gravitropic response of inflorescence stems, (a) the gravity perception site exists through the elongating zone, (b) auxin is involved in this response, (c) the gravitropic curvature was inhibited at 4[deg]C but at least the gravity perception step could occur, and (d) two curvatures could be induced in sequence at 23[deg]C by two opposite directional horizontal gravistimulations at 4[deg]C.

SGR1, SGR2, and SGR3: Novel Genetic Loci Involved in Shoot Gravitropism in Arabidopsis thaliana

In higher plants shoots show a negative gravitropic response but little is known about its mechanism. To elucidate this phenomenon, we have isolated a number of mutants with abnormal shoot gravitropic responses in Arabidopsis thaliana. Here we describe mainly three mutants: sgr1–1, sgr2–1, and sgr3–1 (shoot gravitropism). Genetic analysis confirmed that these mutations were recessive and occurred at three independent loci, named SGR1, SGR2, and SGR3, respectively. In wild type, both inflorescence stems and hypocotyls show negative gravitropic responses. The sgr1–1 mutants showed no response to gravity either by inflorescence stems or by hypocotyls. The sgr2–1 mutants also showed no gravitropic response in inflorescence stems but showed a reduced gravitropic response in hypocotyls. In contrast, the sgr3–1 mutant was found to have reduced gravitropic responses in inflorescence stems but normal gravitropic responses in hypocotyls. These results suggest that some genetic components of the regulatory mechanisms for gravitropic responses are common between inflorescence stems and hypocotyls, but others are not. In addition, these sgr mutants were normal with respect to root gravitropism, and their inflorescence stems and hypocotyls could carry out phototropism. We conclude that SGR1, SGR2, and SGR3 are novel genetic loci specifically involved in the regulatory mechanisms of shoot gravitropism in A. thaliana.

Bacteriophage T3 connector: Three-dimensional structure and comparison with other viral head-tail connecting regions☆

The bacteriophage T3 connector, which consists of 12 copies of protein gp8, has been studied by image processing of electron micrographs from negatively stained ordered aggregates. A three-dimensional reconstruction of T3 connectors was obtained by collection of tilted views and using the direct Fourier method, up to 2.3 nm resolution. The reconstructed unit cell contains two connectors whose main structural features are essentially identical, but facing in opposite directions. The T3 connector has a height of about 10.9 nm, with two clearly defined domains: a wider one 14.4 nm in diameter, with 12 morphological units in the periphery, and a narrower one, 9.7 nm in diameter. There is a channel clearly defined in the narrower domain that almost closes along the wider domain. Comparison of the three-dimensional structure obtained for the connector of phages T3 and phi 29, and that of the neck extracted from phage phi 29 particles, reveals striking similarities and significant differences. A model for a general connector to account for the common functions carried out by these viral assemblies is discussed together with the possible role of the channel for DNA translocation.

Three-dimensional structure of T3 connector purified from overexpressing bacteria☆

The bacteriophage T3 connector has been purified from overexpressed protein in Escherichia coli, harboring a plasmid containing the gene encoding p8 protein. The connector, which is composed of 12 copies of p8, has been crystallized in two-dimensional sheets and studied by electron microscopy from negatively stained specimens. A two-dimensional Fourier filtering and averaging procedure was performed with crystalline specimens. In addition, single particle averaging techniques were used with other preparations. The average images obtained from these two approaches gave similar results. A three-dimensional reconstruction from two-dimensional crystals of T3 connectors was obtained by collecting several sets of tilted views and using standard Fourier procedures. The resolution of the three-dimensional map was 1.65 nm. The reconstructed connector shows two main domains: a wider one with 12 small units in the periphery and with an external diameter of 14.9 nm, and a smaller one with 8.5 nm diameter. The height of the reconstructed connector has been determined to be around 8.5 nm. The reconstruction clearly shows an internal open channel running along the longitudinal axis of the particle and having an average diameter of 3.7 nm.

How do plant shoots bend up? — The initial step to elucidate the molecular mechanisms of shoot gravitropism usingArabidopsis thaliana

In higher plants, shoots show a negative gravitropic response. To elucidate the molecular mechanisms of this phenomenon, mutational analyses usingArabidopsis thaliana are in progress. This minireview aims to present recent developments in the genetic analysis of shoot gravitropism in this organism. We focus mainly on our studies on the novelshootgravitropic (sgr) mutants inArabidopsis thaliana that have dramatic defects in shoot gravitropism.

Structure and assembly of bacteriophage T3 tails

Abstract Bacteriophage T3 gene products found in the virion tail structure are identified by in vitro complementation and serum blocking activity. On the basis of these measurements, a pathway for the assembly of the T3 tail is proposed. The products of genes 11 and 12 (gp11 and gp12) are assembled on the head to form the tail. The assembly of gp11 and gp12 proceeds cooperatively, so that in the absence of either protein, attachment of the other does not occur. T3 serum blocking activity is due to gp17. Gp17 is assembled onto the tail structure after attachment of gpll and gp12 onto the head. The structure and composition of purified T3 tails have been examined. Purified tails have a sixfold longitudinal axis of symmetry. When viewed along the symmetry axis, tails appear hexagonal with a hole in the center and with bent tail fibers radiating from the apices. Tail fiber proteins are controlled by gene 17. Tails isolated from osmotically shocked T3 phage contain gp11, gp12, and gp17. In addition, isolated tails also contain gp8, a minor head protein, suggesting that gp8 is located at a unique site in the T3 head where the tail attaches.

In vitro packaging of phage T3 DNA

Abstract We have developed an in vitro system for packaging of mature bacteriophage DNA. DNA purified from phage T3, when incubated in a reaction mixture containing binary combinations of extracts prepared from Escherichia coli cells infected with T3 amber mutants of genes involved in DNA replication (genes 3, 4, 5, and 6), is packaged intact and fully conserved into infectious particles. The products of genes 3, 4, 5, 6, and 19 (necessary for DNA maturation in vivo ) are required for packaging in vitro . Other requirements are Mg 2 +, spermidine, and either ATP or GTP. The packaging efficiency decreases with decreasing concentration of exogenous DNA. Exogenous DNA was converted into concatemeric forms in the reaction mixture, depending upon the products of genes 3, 4, 5, and 6. These results suggest that the exogenous DNA proceeds by way of concatemeric intermediates before being packaged. Genetic recombination also occurs in the in vitro packaging system. Recombination is accompanied by physical exchange of sequences between exogenous DNA molecules. ATP is required for in vitro recombination; GTP cannot be substituted for ATP in this reaction.

Bacteriophage T3 gene 8 product oligomer structure

The structure of the connector of bacteriophage T3 (built up by the product of gene 8) has been studied in two dimensions by combined use of translational and rotational image filtering procedures applied to tetragonal ordered aggregates of the former oligomers. This analysis, performed up to 1/1.6 nm-1 resolution, has revealed the existence of a 12-fold symmetry in the outermost region of the specimen (mainly between radii 5.2 and 6.7 nm), a 6-fold one in the inner region (between radii 1.7 and 3.2 nm), and a hole in its center. These features are very similar to the ones described for the connectors of other phages, such as T4, lambda, and phi 29, thus suggesting a common mechanism for the functions carried out by this viral region.