Toshihiro Yonekawa

Fission Yeast Eso1p Is Required for Establishing Sister Chromatid Cohesion during S Phase

ABSTRACT Sister chromatid cohesion is essential for cell viability. We have isolated a novel temperature-sensitive lethal mutant namedeso1-H17 that displays spindle assembly checkpoint-dependent mitotic delay and abnormal chromosome segregation. At the permissive temperature, the eso1-H17 mutant shows mild sensitivity to UV irradiation and DNA-damaging chemicals. At the nonpermissive temperature, the mutant is arrested in M phase with a viability loss due to a failure to establish sister chromatid cohesion during S phase. The lethal M-phase arrest phenotype, however, is suppressed by inactivation of a spindle checkpoint. Theeso1+ gene is not essential for the onset and progression of DNA replication but has remarkable genetic interactions with those genes regulating the G1-S transition and DNA replication. The N-terminal two-thirds of Eso1p is highly homologous to DNA polymerase η of budding yeast and humans, and the C-terminal one-third is homologous to budding yeast Eco1p (also called Ctf7p), which is required for the establishment of sister chromatid cohesion. Deletion analysis and determination of the mutation site reveal that the function of the Eco1p/Ctf7p-homologous domain is necessary and sufficient for sister chromatid cohesion. On the other hand, deletion of the DNA polymerase η domain in Eso1p increases sensitivity to UV irradiation. These results indicate that Eso1p plays a dual role during DNA replication. The C-terminal region acts to establish sister chromatid cohesion, and the N-terminal region presumably catalyzes translesion DNA synthesis when template DNA contains lesions that block regular DNA replication.

Development of a new method for diagnosis of rubella virus infection by reverse transcription-loop-mediated isothermal amplification

ABSTRACT We developed a useful method for the detection of rubella virus genome RNA by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and compared the sensitivity of RT-LAMP with that of other virological tests: reverse transcription-PCR (RT-PCR) and virus isolation. The rubella virus genome was amplified by RT-LAMP from clinical isolates obtained between 1987 and 2004 with similar sensitivities to the Takahashi vaccine strain. The detection limit of RT-LAMP was compared with that of RT-PCR using the Takahashi vaccine strain. We detected rubella virus genome material corresponding to 30 PFU/ml in a culture fluid sample by RT-LAMP within 60 min after the extraction of RNA with equal sensitivity to RT-nested PCR. The positive result rates of RT-LAMP, RT-PCR, and virus isolation were also compared using throat swabs obtained from patients who were clinically diagnosed with acute rubella virus infection in 2004 in Tochigi, Japan. Among nine patients with clinical rubella, the positive result rates were three/nine (33.3%) for virus isolation, six/nine (66.7%) for RT-PCR, and seven/nine (77.8%) for RT-LAMP. Consequently, RT-LAMP for rubella virus would be expected to be a reliable rapid diagnostic tool in the clinical setting.

Direct detection of human herpesvirus 6B by the LAMP method using newly developed dry-reagents☆

The reliability of the HHV-6B LAMP using the dry-reagent method was evaluated using serum samples obtained from febrile children. The sensitivity of the original and dry-reagent methods was 10 copies/reaction and 100 copies/reaction, respectively. The dry-reagent LAMP method was highly sensitive (94.0%) and specific (96.0%) for the detection of HHV-6B.