Development of a thermostable Cre/lox-based gene disruption system and in vivo manipulations of the megaplasmid pTT27 in Thermus thermophilus HB27.

We previously reported the development of a Cre/lox-based gene disruption system for multiple markerless gene disruption in Thermus thermophilus; however, it was a time-consuming method because it functioned at 50 °C, the minimum growth temperature of T. thermophilus HB27. In the present study, we improved this system by introducing random mutations into the cre-expressing plasmid, pSH-Cre.

Detection and analysis of UV-induced mutations in the chromosomal DNA of Arabidopsis.

Under natural conditions, plants are exposed to solar ultraviolet (UV) radiation, which damages chromosomal DNA. Although plant responses to UV-induced DNA damage have recently been elucidated in detail, revealing a set of DNA repair mechanisms and translesion synthesis (TLS), limited information is currently available on UV-induced mutations in plants. We previously reported the development of a supF-based system for the detection of a broad spectrum of mutations in the chromosomal DNA of Arabidopsis.

Development of a supF-based mutation-detection system in the extreme thermophile Thermus thermophilus HB27.

Thermus thermophilus (T. thermophilus) HB27 is an extreme thermophile that grows optimally at 65-72 °C. Heat-induced DNA lesions are expected to occur at a higher frequency in the genome of T.

The NAC Transcription Factor Gene OsY37 (ONAC011) Promotes Leaf Senescence and Accelerates Heading Time in Rice.

Leaf senescence is an important physiological process involving the degradation of a number of metabolites and their remobilization to new reproductive and storage organs. NAC (NAM, ATAF, and CUC) transcription factors are reported as important regulators of the senescence process. Here, we describe the identification and functional characterization of the NAC transcription factor gene, (Oryza sativa Yellow37, ONAC011) obtained from cv.

Cre/lox-based multiple markerless gene disruption in the genome of the extreme thermophile Thermus thermophilus.

Markerless gene-disruption technology is particularly useful for effective genetic analyses of Thermus thermophilus (T. thermophilus), which have a limited number of selectable markers. In an attempt to develop a novel system for the markerless disruption of genes in T.

Induction of a dwarf phenotype with IBH1 may enable increased production of plant-made pharmaceuticals in plant factory conditions.

Year-round production in a contained, environmentally controlled 'plant factory' may provide a cost-effective method to produce pharmaceuticals and other high-value products. However, cost-effective production may require substantial modification of the host plant phenotype; for example, using dwarf plants can enable the growth of more plants in a given volume by allowing more plants per shelf and enabling more shelves to be stacked vertically. We show here that the expression of the chimeric repressor for Arabidopsis AtIBH1 (P35S:AtIBH1SRDX) in transgenic tobacco plants (Nicotiana tabacum) induces a dwarf phenotype, with reduced cell size.

Construction of a supF-based system for detection of mutations in the chromosomal DNA of Arabidopsis.

The factors maintaining genomic integrity, which have been studied in detail in other species, have yet to be investigated in plants. Recent progress in gene-silencing technology has made it possible to produce transgenic plants with loss-of-function phenotypes for the effective analysis of these factors, even with the high redundancy of genes in plants. Therefore, a mutation-detection system for plants is necessary to estimate the biological function of a target gene for mutation frequencies and spectra.

Aberrant vegetative and reproductive development by overexpression and lethality by silencing of OsHAP3E in rice.

We generated transgenic rice plants overexpressing OsHAP3E which encodes a subunit of a CCAAT-motif binding HAP complex. The OsHAP3E-overexpressing plants showed various abnormal morphologies both in their vegetative and reproductive phases. The OsHAP3E-overexpressing plants were dwarf with erected leaves and similar to brassinosteroid mutants in the vegetative phase.

The AP2/ERF transcription factor WIND1 controls cell dedifferentiation in Arabidopsis.

Many multicellular organisms have remarkable capability to regenerate new organs after wounding. As a first step of organ regeneration, adult somatic cells often dedifferentiate to reacquire cell proliferation potential, but mechanisms underlying this process remain unknown in plants. Here we show that an AP2/ERF transcription factor, WOUND INDUCED DEDIFFERENTIATION 1 (WIND1), is involved in the control of cell dedifferentiation in Arabidopsis.

Loss of function of the HSFA9 seed longevity program.

Gain of function approaches that have been published by our laboratory determined that HSFA9 (Heat Shock Factor A9) activates a genetic program contributing to seed longevity and to desiccation tolerance in plant embryos. We now evaluate the role(s) of HSFA9 by loss of function using different modified forms of HaHSFA9 (sunflower HSFA9), which were specifically overexpressed in seeds of transgenic tobacco. We used two inactive forms (M1, M2) with deletion or mutation of the transcription activation domain of HaHSFA9, and a third form (M3) with HaHSFA9 converted to a potent active repressor by fusion of the SRDX motif.

The Arabidopsis thaliana STYLISH1 protein acts as a transcriptional activator regulating auxin biosynthesis.

The establishment and maintenance of auxin maxima in vascular plants is regulated by auxin biosynthesis and polar intercellular auxin flow. The disruption of normal auxin biosynthesis in mouse-ear cress (Arabidopsis thaliana) leads to severe abnormalities, suggesting that spatiotemporal regulation of auxin biosynthesis is fundamental for normal growth and development. We have shown previously that the induction of the SHORT-INTERNODES/STYLISH (SHI/STY) family member STY1 results in increased transcript levels of the YUCCA (YUC) family member YUC4 and also higher auxin levels and auxin biosynthesis rates in Arabidopsis seedlings.

Efficient production of male and female sterile plants by expression of a chimeric repressor in Arabidopsis and rice.

Male and female sterile plants are particularly useful for the effective production of commercial hybrid plants and for preventing the diffusion of seeds or pollen grains of genetically modified plants in the open field. In an attempt to create several types of sterile plant by genetic manipulation, we applied our Chimeric REpressor Gene-Silencing Technology (CRES-T) to four transcription factors, namely APETALA3, AGAMOUS, LEAFY and AtMYB26, involved in the regulation of petal and stamen identity, stamen and carpel identity, floral meristem identity and anther dehiscence, respectively, in Arabidopsis. Transgenic plants expressing each chimeric repressor exhibited, at high frequency, a sterile phenotype that resembled the loss-of-function phenotype of each corresponding gene.

AREB1 is a transcription activator of novel ABRE-dependent ABA signaling that enhances drought stress tolerance in Arabidopsis.

ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1) (i.e., ABF2) is a basic domain/leucine zipper transcription factor that binds to the abscisic acid (ABA)-responsive element (ABRE) motif in the promoter region of ABA-inducible genes.

A chimeric AtMYB23 repressor induces hairy roots, elongation of leaves and stems, and inhibition of the deposition of mucilage on seed coats in Arabidopsis.

We reported previously that a chimeric repressor, in which a transcription factor was fused to the EAR motif repression domain, acted as a dominant repressor and suppressed the expression of target genes, such that resultant phenotypes were similar to those associated with loss-of-function alleles. We report here that expression of the chimeric AtMYB23 repressor induced a variety of morphological changes, namely the ectopic formation of root hairs, a short primary root, elongation of leaves and of inflorescence stems, and absence of the accumulation of mucilage on seed coats, in addition to disruption of the development of trichomes. The short primary root and the elongation of leaves and stems appeared to be due to the reduced and enhanced lengthwise expansion, respectively, of epidermal cells.

Identification of the minimal repression domain of SUPERMAN shows that the DLELRL hexapeptide is both necessary and sufficient for repression of transcription in Arabidopsis.

We reported previously that the carboxy-terminal 30 amino acids of SUPERMAN (SUPRD) function as a repression domain in Arabidopsis. In this study, we identified the peptide sequences in SUPRD that is both necessary and sufficient for repression of transcription. To our surprise, the hexapeptide DLELRL was sufficient, by itself, to confer the ability to repress transcription on a DNA-binding domain.

A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway.

Arabidopsis thaliana RD26 cDNA, isolated from dehydrated plants, encodes a NAC protein. Expression of the RD26 gene was induced not only by drought but also by abscisic acid (ABA) and high salinity. The RD26 protein is localized in the nucleus and its C terminal has transcriptional activity.

Circularized chromosome with a large palindromic structure in Streptomyces griseus mutants.

Streptomyces linear chromosomes display various types of rearrangements after telomere deletion, including circularization, arm replacement, and amplification. We analyzed the new chromosomal deletion mutants Streptomyces griseus 301-22-L and 301-22-M. In these mutants, chromosomal arm replacement resulted in long terminal inverted repeats (TIRs) at both ends; different sizes were deleted again and recombined inside the TIRs, resulting in a circular chromosome with an extremely large palindrome.

Chromosomal circularization in Streptomyces griseus by nonhomologous recombination of deletion ends.

Streptomyces linear chromosomes frequently cause deletions at both ends spontaneously or by various mutagenic treatments, and concomitantly display dynamic structural changes such as circularization and arm replacement. We have cloned and sequenced the fusion junctions of circularized chromosomes in two deletion mutants of Streptomyces griseus. No homology and a 1-bp overlap were found between the deletion ends of the mutant chromosomes.

The large linear plasmid pSLA2-L of Streptomyces rochei has an unusually condensed gene organization for secondary metabolism.

The complete nucleotide sequence of the large linear plasmid pSLA2-L in Streptomyces rochei strain 7434AN4 has been determined. pSLA2-L was found to be 210 614 bp long with a GC content of 72.8% and carries 143 open reading frames.

Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis.

The redundancy of genes for plant transcription factors often interferes with efforts to identify the biologic functions of such factors. We show here that four different transcription factors fused to the EAR motif, a repression domain of only 12 amino acids, act as dominant repressors in transgenic Arabidopsis and suppress the expression of specific target genes, even in the presence of the redundant transcription factors, with resultant dominant loss-of-function phenotypes. Chimeric EIN3, CUC1, PAP1, and AtMYB23 repressors that included the EAR motif dominantly suppressed the expression of their target genes and caused insensitivity to ethylene, cup-shaped cotyledons, reduction in the accumulation of anthocyanin, and absence of trichomes, respectively.

Cloning and analysis of the telomere and terminal inverted repeat of the linear chromosome of Streptomyces griseus.

Cloning and sequencing of the telomere of Streptomyces griseus revealed five palindromic sequences in the terminal 116 nucleotides, all of which can make a hairpin loop structure. However, the end sequence cannot form the foldback secondary structure that is common in Streptomyces telomeres and is suggested to be necessary for terminal replication. Both inside ends of the terminal inverted repeat (TIR) were also cloned and sequenced.

The SUPERMAN protein is an active repressor whose carboxy-terminal repression domain is required for the development of normal flowers.

SUPERMAN was identified as a putative regulator of transcription that acts in floral development, but its function remains to be clarified. We demonstrate here that SUPERMAN is an active repressor whose repression domain is located in the carboxy-terminal region. Ectopic expression of SUPERMAN that lacked the repression domain resulted in a phenotype similar to that of superman mutants, demonstrating that the repression activity of SUPERMAN is essential for the development of normal flowers.