Current human leukocyte antigen (HLA) DNA typing methods such as the sequence-based typing (SBT) and sequence-specific oligonucleotide (SSO) methods generally yield ambiguous typing results because of oligonucleotide probe design limitations or phase ambiguity for HLA allele assignment. Here we describe the development and application of the super high-resolution single-molecule sequence-based typing (SS-SBT) of HLA loci at the 8-digit level using next generation sequencing (NGS). NGS which can determine an HLA allele sequence derived from a single DNA molecule is expected to solve the phase ambiguity problem. Eight classical HLA loci-specific polymerase chain reaction (PCR) primers were designed to amplify the entire gene sequences from the enhancer-promoter region to the 3' untranslated region. Phase ambiguities of HLA-A, -B, -C, -DRB1 and -DQB1 were completely resolved and unequivocally assigned without ambiguity to single HLA alleles. Therefore, the SS-SBT method described here is a superior and effective HLA DNA typing method to efficiently detect new HLA alleles and null alleles without ambiguity.

[1] We made middle and upper atmosphere (MU) radar observations of midlatitude E region field-aligned irregularities (FAIs) in the summer of 1999 and 2000. Sporadic E (E s ) layer was monitored with a routine ionosonde, and its altitude was measured with an FM-CW sounder (FCS). In this paper we draw attention to two findings. First, we show that quasiperiodic (QP) radar echoes appearing before 0200 LT are more enhanced with increasing f o E s À f b E s , which means that the FAI generation is closely related to localized density gradients within E s , and extend from 100 to 130 km in altitude, while E s altitudes determined from the FCS soundings are between 100 and 110 km. The latter fact suggests that existing models for the QP echo generation, which require a deep modulation of E s altitude, are not applicable to our observational results. We propose a new working model for generating QP echoes in which polarization electric fields originated from high-density plasma clouds within E s are mapped upward along the geomagnetic field to produce relatively weak irregularities above the E s layer. Second, we show new findings obtained from the current observations, namely, two types of QP echoes that occur below 100 km in the morning: one is the morning QP (MQP) echoes with periods of 4-8 min, and the other is the QP echoes with periods of $1 min. The latter type can be categorized as low-altitude QP echoes that were found from previous nighttime MU radar observations. Until now the MU radar QP echoes have been believed to occur above 100 km for the period from sunset to midnight. Although we do not know the generation mechanisms of the low-altitude MQP echoes, we suppose that these echoes might be caused by a weak E s that exists below 100 km.

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From October 2013 to date, approximately 1,000 outbreaks of porcine epidemic diarrhoea virus (PEDV) have occurred in Japan. Porcine epidemic diarrhoea with non-lethal effects in piglets was identified in Tottori prefecture in October 2014. Complete genome analysis revealed that the causative pathogen, Tottori2, is a new PEDV variant with a large (582 nt) deletion in the spike gene. Phylogenetic analysis indicated that the Tottori2 PEDV strain might have been derived from the current PEDV strains circulating in domestic pigs. Moreover, the Tottori2 PEDV strain was successfully isolated in Vero cells by serial passage.

To investigate cell cycle regulation at the S or G2 phase in Saccharomyces cerevisiae, we have isolated mutants displaying supersensitivity to hydroxyurea (HU), a chemical that inhibits DNA replication. Such mutants, which we have named hydroxyurea sensitive (hys), defined four linkage groups and we characterized the hys2 mutation in this study. The hys2-1 mutant displays temperature sensitive growth and a constellation of phenotypes indicating defective DNA metabolism. At the restrictive temperature, hys2-1 cells arrest as large budded cells with a single nucleus at the neck of the bud and a short spindle. The hys2-1 mutant exhibits increased rates of chromosome loss and recombination. Additionally, hys2-1 appears to accumulate incompletely replicated DNA that can be detected by a pulse field electrophoresis assay. Finally, deletion of RAD9 in a hys2-1 strain decreases the percentage of arrested cells, suggesting that an intact RAD9-checkpoint is required for the cell cycle arrest in hys2-1 cells. HYS2 encodes a 55 kDa protein that is essential for viability at all temperatures. Taken together, these data suggest that Hys2 plays a role in DNA replication.