Keiko Taki

Gene Expression Profiles in Mouse Liver after Long-Term Low-Dose-Rate Irradiation with Gamma Rays

Abstract Changes in gene expression profiles in mouse liver induced by long-term low-dose-rate &ggr; irradiation were examined by microarray analysis. Three groups of male C57BL/6J mice were exposed to whole-body radiation at dose rates of 17–20 mGy/day, 0.86–1.0 mGy/day or 0.042–0.050 mGy/day for 401–485 days with cumulative doses of approximately 8 Gy, 0.4 Gy or 0.02 Gy, respectively. The gene expression levels in the livers of six animals from each exposure group were compared individually with that of pooled sham-irradiated animals. Some genes revealed a large variation in expression levels among individuals within each group, and the number of genes showing common changes in individuals from each group was limited: 20 and 11 genes showed more than 1.5-fold modulation with 17–20 mGy/day and 0.86–1.0 mGy/day, respectively. Three genes showed more than 1.5-fold modulation even at the lowest dose-rate of 0.04–0.05 mGy/day. Most of these genes were down-regulated. RT-PCR analysis confirmed the expression profiles of the majority of these genes. The results indicate that a few genes are modulated in response to very low-dose-rate irradiation. The functional analysis suggests that these genes may influence many processes, including obesity and tumorigenesis.

Construction of a URA5.3 deletion strain of the unicellular red alga Cyanidioschyzon merolae: A backgroundless host strain for transformation experiments

None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work. In this paper, we report the isolation of a mutant in which the URA5.3 gene is completely deleted, and which has been successfully used for backgroundless selection of transformants. It has previously been shown in C. merolae that the nuclear genome can be modified via homologous recombination with exogenously introduced DNA (Fujiwara et al., 2013; Imamura et al., 2009). Here, we isolated a URA5.3 deletion mutant, by transforming with a URA5.3-deleted DNA fragment and directly selecting 5-FOA-resistant clones. The URA5.3-deleted fragment (URA5.3D fragment, Fig. 1a) was generated using three steps of the PCR. The first PCR amplified a chromosomal fragment, from the 5′ upstream position –2708 to +1415, with respect to the URA5.3 ORF initiation site. This used primers A (5′TCTCGGCTGAGAGGCCTGTTCG-3 ′); and B (5 ′cgcttgcttctgcccattaggaattaAGTCTTACAACAGTACTCAGATCGTTG-3′), in which uppercase letters indicate the sequence upstream of the deleted region, underlining indicates the altered base that prevents the occurrence of non-native translation, and lowercase letters indicate the sequence adjacent to the 3′ end of the URA5.3 ORF. Primer B spanned a 2286-bp deleted region in its sequence. This PCR was carried out using KOD-Plus-Neo DNA polymerase, following the manufacturer’s instructions (Toyobo Co., Ltd., Osaka, Japan). Similarly, a fragment from position –924 to +3352 was PCR-amplified by primers C (5′-CAACGATCTGAGTACTGTTGTAAGACTtaattcctaatgggcagaagcaagcg-3′), and D (5′-CTATGTCTGCTTTCACCAGGGTGG-3′). These two fragConstruction of a URA5.3 deletion strain of the unicellular red alga Cyanidioschyzon merolae: A backgroundless host strain for transformation experiments

Involvement of Oct-1 in the regulation of CDKN1A in response to clinically relevant doses of ionizing radiation

CDKN1A is a cyclin-dependent kinase inhibitor that plays a critical role in cell cycle checkpoint regulation. It is transcriptionally induced by TP53 (p53) following exposure to ionizing radiation (IR). Induction of CDKN1A after irradiation is closely related to IR-sensitivity of tumor cells, but the underlying mechanisms remain obscure because conventional reporter gene systems respond poorly to IR unless hyperlethal doses are used. Here, we performed a promoter analysis of the CDKN1A gene following irradiation with clinically relevant doses of IR using the adeno-associated virus-mediated reporter system which we have recently shown to be highly responsive to IR. We demonstrate that there are regulatory elements at -1.1 kb, -1.4 kb, and -1.8 kb, and deletion of these elements attenuate induction of the CDKN1A gene promoter in response to 0.2-2.0 Gy of IR. EMSA and ChIP assays showed that Oct-1 binds constitutively to the elements at -1.1 kb and -1.8 kb. Functional involvement of Oct-1 was confirmed by RNA interference targeting the Oct-1 gene, which suppressed both the basal and IR-inducible components of the CDKN1A expression. Thus, our results reveal that Oct-1 is crucial to the TP53-mediated regulation of the CDKN1A gene promoter following exposure to clinically relevant doses of IR.

Construction of a rapamycin-susceptible strain of the unicellular red alga Cyanidioschyzon merolae for analysis of the target of rapamycin (TOR) function

None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work. 2004). Transformants were then selected using the UMP synthase gene as a marker for uracil prototrophy (Imamura et al., 2013). Using the C. merolae F12 strain, we recently revealed that TOR plays a central role in triacylglycerol accumulation in microalgae (Imamura et al., 2015). Because of its simple cell architecture and genome, low gene redundancy, and capacity for gene-knockout by homologous recombination, C. merolae is considered to be a good model photosynthesizing eukaryote for understanding various fundamental molecular mechanisms (Imamura et al., 2009; Kuroiwa, 1998; Matsuzaki et al., 2004). Although these features also make C. merolae suitable for the analysis of TOR functions using the F12 strain, no selection marker is available in F12 enabling its use as a host. Consequently, the establishment of a novel C. merolae strain having both rapamycin-sensitive and uracilauxotrophic phenotypes is required to further investigate TOR functions using a molecular genetic approach. In a recent study, we demonstrated that the T1 strain, in which the URA5.3 gene is completely deleted, is a 5fluoroorotic acid (5-FOA)-resistant, backgroundless host for transformation experiments (Taki et al., 2015). In the study reported here, we constructed a plasmid pKTL1, serving as a genomic knock-in cassette for insertion into the URA5.3 gene (Fig. 1a), and obtained a novel rapamycin-susceptible C. merolae strain in which the URA5.3 gene is replaced by a DNA fragment for constitutive expression of S. cerevisiae FKBP12 (hereafter referred to as the ScFKBP fragment) (Imamura et al., 2013). We then successfully used the isolated strain as a host strain Construction of a rapamycin-susceptible strain of the unicellular red alga Cyanidioschyzon merolae for analysis of the target of rapamycin (TOR) function

A Case of a Child with an APC Pathogenic Mutation, Aberrant Expression of Splice Variants and Positive Family History of FAP

Familial adenomatous polyposis is an autosomal dominant hereditary disease characterized by the appearance of hundreds to thousands of colorectal adenomatous polyps; if left untreated, there is nearly a 100% lifetime risk of colorectal cancer. In the present case, adenomatous polyps were observed at 6 years of age. Unlike our previous assumption, adenomatous polyps were detected by colonoscopy at <10 years of age. Considering the clinical importance of early diagnosis, we report this case involving germline adenomatous polyposis coli mutation (c.1958G > C, GenBank: M74088.1) that caused an increase in the isoform without exon 15. Although this isoform has been reported previously, it remains controversial whether the variant is pathogenic or not because it was observed both in patients with familial adenomatous polyposis and in normal controls. Nonetheless, due to quantitative distortion of splice variants in adenomatous polyposis coli transcripts and the early development of adenomatous polyps, we believe that this variant may be pathogenic.