Among a large collection of nonsense (termination) suppressors of Saccharomyces cerevisiae, a few remained obscure for their molecular nature. Of those, a group of weak and recessive suppressors, sup111, sup112 and sup113, is of particular interest because of their dependency on [PSI+], a yeast prion. From the facts that these suppressors map at positions quite similar to the UPF2, UPF3 and UPF1 genes, respectively, and that some mutations in the UPF genes confer termination suppressor activity, we suspected that sup111, sup112 and sup113 would very well be mutant alleles of the UPF genes. We tested our speculation and found that sup113, sup111 and sup112 were in fact complemented with the wild-type alleles of UPF1, UPF2 and UPF3, respectively. We further obtained evidence that the UPF1, UPF2 and UPF3 loci of the strains carrying sup113, sup111 and sup112, respectively, had point mutations. From these results, we conclude that sup111, sup112 and sup113 are mutant alleles of UPF2, UPF3 and UPF1, respectively, and thus attribute suppressor activity of these mutations to defects in the NMD (nonsense-mediated mRNA decay) machinery.
A novel sporulation-deficient mutant, sev4-L5, was isolated in a genetic screen of a collection of temperature-sensitive mutants of Schizosaccharomyces pombe. The wild-type sev4 gene was identified as cta4+, which encodes a putative cation-transporting P-type ATPase. The sev4-L5 allele harbored a single missense mutation that caused replacement of Gly615 with a glutamate at the putative ATP-binding site. Similar to cta4-null mutants, sev4-L5 exhibited defects in growth at high and low temperatures, and sensitivity to high and extremely low concentrations of Ca2+. The cta4+ mRNA level was considerably enhanced during meiosis. When sev4-L5 cells were incubated in sporulation medium at the permissive temperature, meiotic nuclear divisions proceeded with normal kinetics, but spores were not formed. Structural alteration of the spindle pole body, which is prerequisite to construction of the forespore membrane in wild type, was incomplete. Consequently, formation of the forespore membrane was severely impaired. These observations show that perturbation of Ca2+ homeostasis by mutation of cta4/sev4 blocks sporulation mainly by interfering with forespore membrane assembly.
It is known that some local anesthetics inhibit the growth of budding yeast cells. To investigate the pathway of local anesthetics’ action, we isolated and characterized mutants that were hyper-sensitive to tetracaine, and at the same time, temperature-sensitive for growth. They were collectively called las (local anesthetic sensitive) mutants. One of the LAS genes, LAS24, was found to be identical to KOG1, which had been independently discovered as a member of the TOR complex 1 (TORC1). Las24p/Kog1p is a widely conserved TOR binding protein containing the NRC domain, HEAT repeats and WD-40 repeats, but its function remains unknown. Like the tor mutants, the las24 mutants were found to have a defect in cell wall integrity and to show sensitivity to rapamycin. Furthermore, Las24p is required not only in TORC1-mediated (rapamycin-sensitive) pathways such as translation initiation control and phosphorylation of Npr1p and Gln3p, but also for the normal distribution of the actin cytoskeleton, which has been regarded as a TORC2-mediated event. Intriguingly, the temperature-sensitivity of the las24 mutant was suppressed by either activation of Tap42/PPase or by down-regulation of the RAS/cAMP pathway. Suppressors of the temperature-sensitivity of the las24-1 mutant were found not to be effective for suppression of the tetracaine-sensitivity of the same mutant. These observations along with the facts that tetracaine and high temperature differentially affected the las24-1 mutant suggest that Las24p/Kog1p is not a target of tetracaine and that the tetracaine-sensitive step may be one of downstream branches of the TORC1 pathway. Consistent with the broad cellular functions exerted by the TOR pathway, we found that Las24p was associated with membranes and was localized at vacuoles, the plasma membrane and small vesicles.
The variations in nuclear DNA content from 2 × 102 to 2.5 × 105 Mbp are reported in higher plants. The major finding so far is that the genome size of plant species differs by three orders of magnitude, which are more variable than the other organisms. Investigations pertaining to the manner in which DNA is packaged in the nucleus provide us with basic information on the made of DNA existence in the plant nucleus. However, the fundamentals on nuclear DNA content and nuclear size, which underlie and enable the flexible containment of such large differences in nuclear DNA content, remain unknown. We analyzed the nuclear volumes of plants with 2C value DNA contents ranging from 3.2 × 102 to 1.0 × 105 Mbp. As a result, we obtained a constant ratio between the DNA volume and nuclear volume, which does not exceed 3%. Furthermore, we also demonstrate that the nuclear Rabl model of chromatin organisation is not a common 3-D structure, even in plants with large nuclear DNA contents. The existence of an upper limit of DNA volume ratio would present a basal parameter for the future insight into the nuclear organisation in higher plants.
During the “Green Revolution” of rice, high-yielding varieties (HYVs) were developed using a semi-dwarf gene (sd1 or OsGA20ox2). The presence or absence of the two mutant alleles (DGWG type in Dee-geo-woo-gen and JKK type in Jikkoku) were surveyed by PCR using 256 accessions of eight wild and two cultivate rice species. The DGWG allele was detected in a landrace (Oryza sativa) and two accessions of wild rice (O. rufipogon), all of which are from China, showing their limited distribution. Genealogical studies of the OsGA20ox2 gene showed that the 62 sequences of O. sativa and O. rufipogon included 20 distinct haplotypes, indicating that the species complex contained OsGA20ox2 genes from two different lineages. The silent site nucleotide diversities (π and θw) were extremely low in Japonica rice, suggesting a genetic bottleneck. The haplotype network showed that the DGWG and JKK alleles were derived in different lineages. The DGWG carrier (W1944) had unique polymorphisms in the surrounding region of the locus, suggesting that the DGWG allele has been preserved in the wild progenitor, rather than that the DGWG allele has been introgressed from HYVs to W1944. Although a semi-dwarfing plant is a weak competitor under saturated fields, the crossing experiment revealed that the DGWG variant might have been preserved as a hidden variation in the genetic background of wild rice, without expressing a short-stature.
From about 10000 PCR-based EST markers of barley we chose 1421 EST markers that were demonstrated to be amplified differently by PCR between wheat (Triticum aestivum cv. Chinese Spring) and barley (Hordeum vulgare cv. Betzes). We assigned them to the seven barley chromosomes (1H to 7H) by PCR analysis using a set of wheat-barley chromosome addition lines. We successfully assigned 701 (49.3%) EST markers to the barley chromosomes: 75 to 1H, 127 to 2H, 119 to 3H, 94 to 4H, 108 to 5H, 81 to 6H and 97 to 7H. By using a set of Betzes barley telosomic addition lines of Chinese Spring, we could successfully determine the chromosome-arm (S or L) location of at least 90% of the EST markers assigned to each barley chromosome. We conducted a trial mapping using 90 EST markers assigned to 7HS (49) or 7HL (41) and 19 wheat lines carrying 7H structural changes. More EST markers were found in the distal region than in the proximal region.
We conducted a pilot survey of genetic diversity among 37 karyotyped individuals of the black rat Rattus rattus (sensu lato) from six localities on the Japanese Islands, using complete gene sequences of mitochondrial cytochrome b (cyt b) and nuclear interphotoreceptor retinoid binding protein (IRBP). Our sampling included two previously documented karyotypic groups: ‘Oceanian’ with 2n = 38 and ‘Asian’ with 2n = 42. Cyt b sequences for most individuals clustered according to their karyotypic groups, with an average between-group divergence of 3.8%. One exception was that individuals from Kagoshima (Kyushu Island) showed ‘Asian’ karyotypes combined with a cyt b haplotype that differed by a single nucleotide substitution from the haplotype of the ‘Oceanian’ karyotypic group. Six IRBP haplotypes were identified. They belonged to three distinct IRBP lineages (I-III), with an average inter-lineage divergence of 1%. Among homozygous individuals, these lineages showed good association with the karyotypic groups: IRBP lineage I occurred only with ‘Oceanian’ karyotypes, while IRBP lineages II and III both occurred with ‘Asian’ karyotypes. Individuals from Kagoshima all possessed IRBP of ‘Asian’ lineages, despite the presence of an ‘Oceanian’ mitochondrial type. The Chichijima population (Ogasawara Islands) featured exclusively ‘Asian’ karyotypes and cyt b sequences, but various combinations of all three IRBP lineages. The Kagoshima and Chichijima populations thus provide strong evidence of viable hybridization and genetic introgression between the two karyotypic groups, but with variable genetic outcomes. Our results demonstrate the potential of combined analysis of karyotypes and mitochondrial and nuclear gene sequences to elucidate the complex dispersal and population history of the black rat.
Gene 1 product (gp1) of Bacillussubtilis phage φ29 is known to promote DNA replication of the phage. Although its role in the DNA replication is not clear, gp1 is reported to exhibit multiple characteristics, including RNA binding, cell membrane localization, and self-association. To investigate these characteristics, we undertook the isolation of a series of missense mutants of gene 1 bearing substitutions at various regions. During cloning of gene 1, we found that its expression severely inhibited the growth of its host Escherichiacoli cells. In this study, we utilized this growth-inhibition phenomenon to screen a random library muta-genized by error-prone PCR, expecting that mutants which could not inhibit cell growth would be affected in the authentic functions of gp1. Using this approach, we obtained 31 different mutants bearing single amino acid substitutions at 26 positions along the entire length of gp1. As a preliminary analysis of these mutants, we compared the deduced amino acid sequences of gp1s from φ29 and its related phages PZA, B103 and M2. Alignment of these sequences revealed three conserved regions, i.e. a hydrophobic region near the carboxyl terminus (assumed to be involved in the membrane localization and self-association of gp1), coiled-coil motif (essential for self-association), and a region of unknown function near the amino terminus. Interestingly, many of the substitutions in the isolated mutants occurred at strongly conserved residues in these regions and affected characteristic features of the regions (e.g. hydrophobicity of the hydrophobic region). These substitutions are expected to affect authentic functions of gp1, and the mutants will be useful for studies of the structure and functions of gp1.