BackgroundHeavy-ion mutagenesis is recognised as a powerful technology to generate new mutants, especially in higher plants. Heavy-ion beams show high linear energy transfer (LET) and thus more effectively induce DNA double-strand breaks than other mutagenic techniques. Previously, we determined the most effective heavy-ion LET (LETmax: 30.0 keV μm-1) for Arabidopsis mutagenesis by analysing the effect of LET on mutation induction. However, the molecular structure of mutated DNA induced by heavy ions with LETmax remains unclear. Knowledge of the structure of mutated DNA will contribute to the effective exploitation of heavy-ion beam mutagenesis.ResultsDry Arabidopsis thaliana seeds were irradiated with carbon (C) ions with LETmax at a dose of 400 Gy and with LET of 22.5 keV μm-1 at doses of 250 Gy or 450 Gy. The effects on mutation frequency and alteration of DNA structure were compared. To characterise the structure of mutated DNA, we screened the well-characterised mutants elongated hypocotyls (hy) and glabrous (gl) and identified mutated DNA among the resulting mutants by high-resolution melting curve, PCR and sequencing analyses. The mutation frequency induced by C ions with LETmax was two-fold higher than that with 22.5 keV μm-1 and similar to the mutation frequency previously induced by ethyl methane sulfonate. We identified the structure of 22 mutated DNAs. Over 80% of the mutations caused by C ions with both LETs were base substitutions or deletions/insertions of less than 100 bp. The other mutations involved large rearrangements.ConclusionsThe C ions with LETmax showed high mutation efficiency and predominantly induced base substitutions or small deletions/insertions, most of which were null mutations. These small alterations can be determined by single-nucleotide polymorphism (SNP) detection systems. Therefore, C ions with LETmax might be useful as a highly efficient reverse genetic system in conjunction with SNP detection systems, and will be beneficial for forward genetics and plant breeding.

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We examined mRNA expression and internal tandem duplication of the Fms-like tyrosine kinase 3 (FLT3) gene in haematological malignancies by reverse transcriptase-polymerase chain reaction (RT-PCR) and genomic PCR followed by sequencing. By RT-PCR, expression of FLT3 was detected in 45/74 (61%) leukaemia cell lines and the frequency of expression of FLT3 was significantly higher in undifferentiated type (B-precursor acute lymphoblastic leukaemia; ALL) than in differentiated type cell lines (B-ALL) (P = 0.0076). Using the genomic PCR method, 194 fresh samples including 87 acute myeloid leukaemias, 60 ALLs, 32 myelodysplastic syndromes (MDSs) and 15 juvenile chronic myelogenous leukaemias (JCMLs) were examined. Tandem duplication was found in 12 (13.8%) AMLs and two (3.3%) ALLs. Sequence analyses of the 14 samples with the duplication revealed that eight showed a simple tandem duplication and six a tandem duplication with insertion. Most of these tandem duplications occurred within exon 11, and two duplications occurred from exon 11 to intron 11 and exon 12. No tandem duplications of FLT3 gene were detected in MDS or JCML. The frequency of tandem duplication of FLT3 gene in childhood AML was lower than that in adult AML so far reported. All of the 12 AML patients with the duplication died within 47 months after diagnosis, whereas two ALL patients with the duplication have survived 44 and 72 months, respectively. These two ALL patients expressed both lymphoid and myeloid antigens and were considered to have biphenotypic leukaemia. These results suggest that tandem duplication is involved in ALL in addition to AML, but not in childhood MDS or JCML, and that childhood AML patients with the tandem duplication have a poor prognosis.

Vascular endothelial cells (EC) exhibit organ-to-organ heterogeneity in their functions and morphologies. In particular, brain capillary EC have unique characteristics exemplified by the blood-brain barrier (BBB). The formation and the maintenance of BBB have been ascribed to EC responses to inductive signal(s) or factor(s) from astrocytes that encircle microvessels in the central nervous system. These EC responses were demonstrated in numerous in vivo studies, exemplified by those of Janzer and Raff (Nature 325:253, 1987) and Tout et al. (Neuroscience 55:291, 1993) showing that transplanted astrocytes induced BBB properties in non-neural vascular EC. In this study, we constructed a heterologous co-culture system, in which rat fetal brain astrocytes were cultivated on one surface of a porous membrane and human umbilical vein EC on the opposite surface. Electron microscopic examination revealed that astrocytes passed their endfeet through the pores, making contact with EC. In this system, gamma-glutamyltranspeptidase (gamma-GTP) activity in EC was found to be significantly increased by contacting astrocytes in a density- and time-dependent manner, but not when the astrocyte feeder layer was apart from EC or replaced by COS cells; astrocyte-derived extracellular matrix partially activated gamma-GTP. mRNAs for some of the representative BBB markers, including transferrin receptor, P-glycoprotein, brain-type glucose transporter (GLUT-1), and gamma-GTP were also demonstrated by reverse transcription-polymerase chain reaction to be upregulated in EC co-cultured with astrocytes. Astrocyte inductions of close membrane apposition resembling a zonula occludens and of an increase in the content of mitochondria in EC were also noted in electron micrographs. Furthermore, an increased barrier activity against inulin was conferred on EC when they were lined with astrocytes. The results obtained with this heterologous co-culture system thus indicate that through contact with their feet, astrocytes are capable of transdifferentiating non-neural EC into the brain type, endowing them with the BBB properties.

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The different forms of flowers in a species have attracted the attention of many evolutionary biologists, including Charles Darwin. In Fagopyrum esculentum (common buckwheat), the occurrence of dimorphic flowers, namely short-styled and long-styled flowers, is associated with a type of self-incompatibility (SI) called heteromorphic SI. The floral morphology and intra-morph incompatibility are both determined by a single genetic locus named the S-locus. Plants with short-styled flowers are heterozygous (S/s) and plants with long-styled flowers are homozygous recessive (s/s) at the S-locus. Despite recent progress in our understanding of the molecular basis of flower development and plant SI systems, the molecular mechanisms underlying heteromorphic SI remain unresolved. By examining differentially expressed genes from the styles of the two floral morphs, we identified a gene that is expressed only in short-styled plants. The novel gene identified was completely linked to the S-locus in a linkage analysis of 1,373 plants and had homology to EARLY FLOWERING 3. We named this gene S-LOCUS EARLY FLOWERING 3 (S-ELF3). In an ion-beam-induced mutant that harbored a deletion in the genomic region spanning S-ELF3, a phenotype shift from short-styled flowers to long-styled flowers was observed. Furthermore, S-ELF3 was present in the genome of short-styled plants and absent from that of long-styled plants both in world-wide landraces of buckwheat and in two distantly related Fagopyrum species that exhibit heteromorphic SI. Moreover, independent disruptions of S-ELF3 were detected in a recently emerged self-compatible Fagopyrum species and a self-compatible line of buckwheat. The nonessential role of S-ELF3 in the survival of individuals and the prolonged evolutionary presence only in the genomes of short-styled plants exhibiting heteromorphic SI suggests that S-ELF3 is a suitable candidate gene for the control of the short-styled phenotype of buckwheat plants.

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