Effect of high-LET Fe-ion beam irradiation on mutation induction in Arabidopsis thaliana.

Heavy-ion beams are powerful mutagens. They cause a broad spectrum of mutation phenotypes with high efficiency even at low irradiation doses and short irradiation times. These mutagenic effects are due to dense ionisation in a localised region along the ion particle path. Linear energy transfer (LET; keV·µm(-1)), which represents the degree of locally deposited energy, is an important parameter in heavy-ion mutagenesis. For high LET radiation above 290 keV∙µm(-1), however, neither the mutation frequency nor the molecular nature of the mutations has been fully characterised. In this study, we investigated the effect of Fe-ion beams with an LET of 640 keV∙µm(-1) on both the mutation frequency and the molecular nature of the mutations. Screening of well-characterised mutants (hy and gl) revealed that the mutation frequency was lower than any other ion species with low LET. We investigated the resulting mutations in the 4 identified mutants. Three mutants were examined by employing PCR-based methods, one of which had 2-bp deletion, another had 178 bp of tandemly duplication, and other one had complicated chromosomal rearrangements with variable deletions in size at breakpoints. We also detected large deletions in the other mutant by using array comparative genomic hybridisation. From the results of the analysis of the breakpoints and junctions of the detected deletions, it was revealed that the mutants harboured chromosomal rearrangements in their genomes. These results indicate that Fe-ion irradiation tends to cause complex mutations with low efficiency. We conclude that Fe-ion irradiation could be useful for inducing chromosomal rearrangements or large deletions.

SlWUS1; an X-linked gene having no homologous Y-linked copy in Silene latifolia.

The dioecious plant Silene latifolia has heteromorphic sex chromosomes, and comparison of the positions of sex-linked genes indicates that at least three large inversions have occurred during the evolution of the Y chromosome. In this article, we describe the isolation of a new sex-linked gene from S. latifolia, which provides new information on the evolution of this plant's young sex chromosomes. By using reverse-transcription polymerase chain reaction degenerate primers based on the Arabidopsis thaliana sequence of WUSCHEL, a flower-development gene, we found two copies in S. latifolia, which we named SlWUS1 and SlWUS2. Southern blot and genetic segregation analysis showed that SlWUS1 is located on the X chromosome and SlWUS2 is autosomal. No Y-linked copy of SlWUS1 was found by either Southern blot analysis under low-stringency conditions or polymerase chain reaction with degenerate primers, so we conclude that SlWUS1 probably has no Y-linked homolog. It is unknown whether the Y chromosome lost the SlWUS1 copy by degeneration of this individual gene or whether deletion of a larger genome region was involved. Several tests lead us to conclude that dosage compensation has not evolved for this sex-linked gene. We mapped the ortholog in the nondioecious relative S. vulgaris (SvWUS1), to compare the location in a species that has no history of having sex chromosomes. SvWUS1 maps to the same linkage group as other fully X-linked genes, indicating that it was not added to the X, but was lost from the Y. Its location differs in the maps from the two species, raising the possibility that the X chromosome, as well as the Y, may have been rearranged.

Two separate pathways including SlCLV1, SlSTM and SlCUC that control carpel development in a bisexual mutant of Silene latifolia.

Carpel suppression is a trigger for sexual dimorphism in the dioecious plant Silene latifolia. To clarify what kind of genes are involved in carpel suppression in this species, we generated a bisexual mutant, R025, by C-ion beam irradiation. R025 produces bisexual flowers with a mature gynoecium and mature stamens. Genetic analysis of R025 attributed the bisexual trait to mutations on the Y chromosome. Scanning electron microscopy (SEM) analysis of early floral development revealed that the carpel size of R025 was different from that of wild-type males in spite of the male background in R025. We also identified an S. latifolia CLAVATA1-like gene (SlCLV1) as a candidate of the CLAVATA-WUSCHEL (CLV-WUS) pathway. Two separate pathways, the CLV-WUS pathway and the CUP-SHAPED COTYLEDON (CUC)-SHOOT MERISTEMLESS (STM) pathway, contribute to carpel development in the Arabidopsis floral meristem. SlSTM1 and SlSTM2 (orthologs of STM) and SlCUC (an ortholog of CUC1 and CUC2) have already been identified in S. latifolia. We therefore examined the expression patterns of SlCLV1, SlSTM (SlSTM1 and SlSTM2) and SlCUC in young flowers of R025 and wild-type males and females. The expression patterns of the three genes in the two pathways differ between the wild-type male and the bisexual mutant, and the differences in expression patterns of the three genes occur at the same stage. These results suggest that in addition to SlSTM1, SlSTM2 and SlCUC, SlCLV1 is also involved in carpel suppression in S. latifolia. They also suggest that a gynoecium-suppressing factor (GSF), which is lost in the R025 Y chromosome, acts on an upstream gene that is common to the two pathways, triggering sexual dimorphism in S. latifolia.

A SUPERMAN-like gene is exclusively expressed in female flowers of the dioecious plant Silene latifolia.

To elucidate the mechanism(s) underlying dioecious flower development, the present study analyzed a SUPERMAN (SUP) homolog, SlSUP, which was identified in Silene latifolia. The sex of this plant is determined by heteromorphic X and Y sex chromosomes. It was revealed that SlSUP is a single-copy autosomal gene expressed exclusively in female flowers. Introduction of a genomic copy of SlSUP into the Arabidopsis thaliana sup (sup-2) mutant complemented the excess-stamen and infertile phenotypes of sup-2, and the overexpression of SlSUP in transgenic Arabidopsis plants resulted in reduced stamen numbers as well as the suppression of petal elongation. During the development of the female flower in S. latifolia, the expression of SlSUP is first detectable in whorls 2 and 3 when the normal expression pattern of the B-class flowering genes was already established and persisted in the stamen primordia until the ovule had matured completely. In addition, significant expression of SlSUP was detected in the ovules, suggestive of the involvement of this gene in ovule development. Furthermore, it was revealed that the de-suppression of stamen development by infection of the S. latifolia female flower with Microbotryum violaceum was accompanied by a significant reduction in SlSUP transcript levels in the induced organs. Taken together, these results demonstrate that SlSUP is a female flower-specific gene and suggest that SlSUP has a positive role in the female flower developmental pathways of S. latifolia.

Floral development of an asexual and female-like mutant carrying two deletions in gynoecium-suppressing and stamen-promoting functional regions on the Y chromosome of the dioecious plant Silene latifolia.

Sexual dimorphism is controlled by genes on the Y chromosome in the dioecious plant Silene latifolia. K034 is the first mutant with female flowers and asexual flowers in one individual. Its stamens are suppressed completely, and its gynoecium exhibits two suppression patterns. One gynoecium resembles a thin rod, as in wild-type males (asexual flower); the other is imperfectly suppressed, having 1-3 carpels (female-like flower). The ratio of these patterns was 9 : 1. To exclude the possibility of chimerism in K034, we crossed a female-like flower of K034 with a wild-type male. Progeny obtained from this crossing had asexual and female-like flowers in one individual. This two-flower-type phenotype was inherited without separating. To examine the identity of flower organs in K034, we analyzed the development of asexual and female-like flowers using scanning electron microscopy and in situ hybridization with SLM1 and SLM2 (orthologs of AGAMOUS and PISTILLATA, respectively) as probes. Mitotic spreads of root tip chromosomes from hairy root cultures showed that K034 had 25 chromosomes. Fluorescent in situ hybridization analysis, using a subtelomeric repetitive sequence (KpnI subfamily) as a probe, indicated that K034 possessed two X chromosomes and one Y chromosome (Y(d)), of which Y(d) had been rearranged to lose the pseudoautosomal region (PAR). PCR analysis using Y-specific sequence-tagged site (STS) markers clarified that Y(d) of K034 had two other deletions in gynoecium-suppressing and stamen-promoting regions. It is reasonable to suggest that these sex chromosomal abnormalities resulted in two abnormal sexual phenotypes: the asexual and imperfect female (female-like) flowers in K034.

The assembly pathway of the 19S regulatory particle of the yeast 26S proteasome.

The 26S proteasome consists of the 20S proteasome (core particle) and the 19S regulatory particle made of the base and lid substructures, and it is mainly localized in the nucleus in yeast. To examine how and where this huge enzyme complex is assembled, we performed biochemical and microscopic characterization of proteasomes produced in two lid mutants, rpn5-1 and rpn7-3, and a base mutant DeltaN rpn2, of the yeast Saccharomyces cerevisiae. We found that, although lid formation was abolished in rpn5-1 mutant cells at the restrictive temperature, an apparently intact base was produced and localized in the nucleus. In contrast, in DeltaN rpn2 cells, a free lid was formed and localized in the nucleus even at the restrictive temperature. These results indicate that the modules of the 26S proteasome, namely, the core particle, base, and lid, can be formed and imported into the nucleus independently of each other. Based on these observations, we propose a model for the assembly process of the yeast 26S proteasome.

[A case of subarachnoid hemorrhage associated with spontaneous intracranial hypotension].

Cerebrospinal fluid (CSF) leakage has recently been documented in most patients, as the main cause of spontaneous intracranial hypotension (SIH). No cases of SIH accompanying an episode of subarachnoid hemorrhage (SAH) has yet been reported and, to the best of our knowledge, this is the first report of a patient, who developed SAH during his clinical course of SIH. SAH was cured completely by surgical intervention. A 57-year-old man, with a history of a sustained postural headache, suffered the sudden onset of severe nuchal pain. SAH in the basal cistern, together with the finding of bilateral subdural fluid retention were verified by CT. Angiogram showed no vascular abnormalities of cerebral or cervical vessels. Gd-enhanced Magnetic Resonance Imaging (MRI) disclosed diffuse meningeal enhancement. RI cisternogram and CT-myelogram (CTM) suggested that the region of CSF leakage should be in the retrospinal area at the level of C1-2. Because orthostatic headache failed to resolve even with 3 weeks of bed rest, surgical intervention was successfully carried out to seal the site of CSF leakage with the use of a fascia and fibrin glue. We have speculated that the etiology of SAH might have been a secondary rupture of congestive intracranial veins, such as basilar plexus or bridging veins, induced by a decrease of intracranial CSF pressure.