Chromosomal DNA replication is a fundamental process in the transmission of genetic information through generations. While the molecular mechanism of DNA replication has been studied for a long time, knowledge regarding this process in eukaryotic cells has advanced rapidly in the past 20 years. Yeast genetics contributed profoundly to this rapid advancement. Reverse genetics and genetic screenings identified all genes encoding replication proteins in budding yeast. Moreover, the genetic interactions that were used in screenings and analyses provided an insight into the molecular mechanism of chromosomal DNA replication. Further studies showed that complicated but sophisticated mechanisms govern chromosomal DNA replication. The retrospective view of the genetic approaches used to elucidate DNA replication in eukaryotes, together with current knowledge, tell us the reasons why some of the genetic screenings are successful, and also provide ideas for future directions.

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Ribosomes translating mRNA without an in-frame stop codon (non-stop mRNA) stall at its 3' end. In eubacteria, such ribosomes are rescued by SsrA-mediated trans-translation. Recently, we have shown that Escherichia coli ArfA (formerly YhdL) also rescues stalled ribosomes by a mechanism distinct from that of trans-translation. Synthetic lethality phenotype of ssrA arfA double mutants suggests that accumulation of stalled ribosomes is deleterious to E. coli cells. In this report, we show that the expression of ArfA is tightly regulated by the system involving trans-translation. Both premature transcription termination and specific cleavage by RNase III were programmed at the specific sites within the arfA open reading frame (ORF) and produced arfA non-stop mRNA. C-terminally truncated ArfA protein synthesized from arfA non-stop mRNA was tagged through SsrA-mediated trans-translation and degraded in wild type cell. In the absence of SsrA, however, C-terminally truncated ArfA escaped from degradation and had a function to rescue stalled ribosomes. Full-length ArfA produced only when arfA mRNA escapes from both premature transcription termination and RNase III cleavage was unstable. From these results, we illustrate a regulatory model in which ArfA is expressed only when it is needed, namely, when the ribosome rescue activity of trans-translation system is insufficient to support cell viability. This sophisticated regulatory mechanism suggests that the ArfA-mediated ribosome rescue is a backup system for trans-translation.

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The experiment on individual chromosome assignments and chromosomal diversity was conducted using a multi-probe fluorescence in situ hybridization (FISH) system in D subgenome of tetraploid Gossypium barbadense (D_b), G. thurberi (D₁) and G. trilobum (D₈), which the later two were the possible subgenome donors of tetraploid cottons. The FISH probes contained a set of bacterial artificial chromosome (BAC) clones specific to 13 individual chromosomes from D subgenome of G. hirsutum (D_h), a D genome centromere-specific BAC clone 150D24, 45S and 5S ribosomal DNA (rDNA) clones, respectively. All tested chromosome orientations were confirmed by the centromere-specific BAC probe. In D₁ and D₈, four 45S rDNA loci were found assigning at the end of the short arm of chromosomes 03, 07, 09 and 11, while one 5S rDNA locus was successfully marked at pericentromeric region of the short arm of chromosome 09. In D_b, three 45S rDNA loci and two 5S rDNA loci were found out. Among them, two 45S rDNA loci were located at the terminal of the short arm of chromosomes D_b07 and D_b09, whilst one 5S rDNA locus was found situating near centromeric region of the short arm of chromosome D_b09. The positions of the BAC clones specific to the 13 individual chromosomes from D_h were compared between D₁, D₈ and D_b. The result showed the existence of chromosomal collinearity within D₁ and D₈, and as well between them and D_b. The results will serve as a base for understanding chromosome structure of cotton and polyploidy evolution of cotton genome and will provide bio-information for assembling the sequences of finished and the on-going cotton whole genome sequencing projects.

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During the domestication of rice (Oryza sativa L.), diversification of flowering time was important in expanding the areas of cultivation. Rice is a facultative short day (SD) plant and requires certain periods of dark to induce flowering. Heading date 1 (Hd1), a regulator of the florigen gene Hd3a, is one of the main factors used to generate diversity in flowering. Loss-of-function alleles of Hd1 are common in cultivated rice and cause the diversity of flowering time. However, it is unclear how these functional nucleotide polymorphisms of Hd1 accumulated in the course of evolution. Nucleotide polymorphisms within Hd1 and Hd3a were analyzed in 38 accessions of ancestral wild rice Oryza rufipogon and compared with those of cultivated rice. In contrast to cultivated rice, no nucleotide changes affecting Hd1 function were found in 38 accessions of wild rice ancestors. No functional changes were found in Hd3a in either cultivated or ancestral rice. A phylogenetic analysis indicated that evolution of the Hd1 alleles may have occurred independently in cultivars descended from various accessions of ancestral rice. The non-functional Hd1 alleles found in cultivated rice may be selected during domestication, because they were not found or very rare in wild ancestral rice. In contrast with Hd3a, which has been highly conserved, Hd1 may have undergone human selection to diversify the flowering times of rice during domestication or the early stage of the cultivation period.

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Vernalization promotes flowering in winter wheat cultivars, whereas spring wheat cultivars are able to transition from vegetative to reproductive phase without vernalization. The wheat vernalization requirement is mainly controlled by the major locus Vrn-1, an APETALA1/FRUITFULL MADS-box gene homolog. To study natural variation of the vernalization requirement in a wild progenitor of common wheat, we sequenced the Vrn-D^t1 locus in four accessions of Aegilops tauschii Coss. Some structural mutations were found in the promoter and first intron regions of Vrn-D^t1, and haplotype analysis was conducted to examine the distribution of each identified mutation within 211 accessions of Ae. tauschii germplasm. Out of the total, nine accessions, which were originally collected in Afghanistan and Pakistan, contained deletions of a 5.4-kb sequence in the critical region of the Vrn-D^t1 first intron. The 5.4-kb deletion mutation appeared independently of the dominant allele of the common wheat Vrn-D1 locus. The large deletion was absolutely associated with a lack of vernalization requirement for flowering under long-day conditions, but had no influence on heading date under field growth conditions. The levels of Vrn-1 and WFT transcript increased in the Ae. tauschii accessions having the large deletion. Identification of natural mutant accessions with a loss of vernalization requirement indicates the agricultural significance of Ae. tauschii as a genetic resource for wheat breeding.

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A total of 2,970 EST–SSRs (2.38%) were identified by transcriptome sequencing of clam Meretrix meretrix (751,970 reads, ~310.82 Mbp), using 454 Genome Sequencer FLX next-generation sequencing platform. Dinucleotide SSR was the dominant repeat type (40.2%), followed by trinucleotide (37.8%), tetranuleotide (12.0%) and pentanucleotide (2.0%) SSR. The dominant repeat motif was AT (71.3%) in the dinucleotide SSR type and AAC (45.6%) in the trinucleotide SSR type. Nearly 79% of all microsatellites had flanking sequences suitable for PCR primer design. Half of PAL were found to be polymorphic in a subset of 40 primer pairs randomly selected. Specifically, the density of dinucleotide, trinucleotide and tetranucleotide repeats showed significant variation among four development stages (trochophore, D-veliger, pediveliger and postlarva). The results suggested that dinucleotide, trinucleotide and tetranucleotide SSRs may play an important role in contributing to the different expression profiles in larval stages.

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An asymptotic approximation of the density function of 2-locus 2-allele model with mutual neutral mutations was obtained invoking the small disturbance asymptotic theory. It was shown by comparing the approximate formula with simulations that this asymptotic method gives a good approximation over the whole time evolution when the mutation rates are high, though it does not give good approximations near the stationary state when the mutation rates are low. On the stationary state, the squared standard linkage deviation made up by using the approximate formula was compared with the exact one obtained by Ohta and Kimura (1969b). It gave a good approximation when the recombination rate is high, even under low mutation rates. Furthermore, as an application of the asymptotic method, The Ancestral Recombination Graph (ARG) was considered.

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As a useful tool to elucidate gene functions, a rice transposon tagging line has been developed using an active endogenous DNA transposon, nDart1. It was highly desirable to evaluate the transposition timing and frequency of the nDart1 elements during rice development to facilitate the generation of an efficient mutant isolation system. Comparison of the detected new insertions at different stages of rice development by transposon display analysis demonstrated that the last heading tiller carry a higher number of nDart1 elements than the main culm. Moreover, it was revealed that the last heading tiller could produce progeny that carried more new insertions of nDart1 elements, mainly as a result of the accumulation of somatic insertions in the parental plant. This report demonstrates that late tillers increase the probability of producing independent mutant lines.

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