In eukaryotes, together with the Mre11/Rad50/Xrs2 (or Nbs1) complex, a family of related protein kinases (the ATM family) is involved in checkpoint activation in response to DNA double-strand breaks. In Saccharomyces cerevisiae, two members of this family, MEC1 and TEL1, have functionally redundant roles in DNA damage repair. Strains with mutations in their mec1 as well as mre11 genes are very sensitive to DNA damaging agents, show defective induction of damage-induced cell-cycle checkpoints, and defective damage-induced homologous recombination. However, the fact that both the mec1Δ and mre11Δ strains exhibit the spontaneous hyper-recombination phenotype is paradoxical in light of the homologous recombination defects in these strains. In this study, we constructed yeast mec1, tel1, and mre11 null mutations and characterized their genome stability properties. Spontaneous and methylmethane sulfonate (MMS)-induced point mutations, base-substitutions, and frameshifts occurred to an almost equal extent in the wild-type, mec1Δ, tel1Δ, and mre11Δ strains. Thus, Mec1, Tel1, and Mre11 do not play roles in the point mutation response. We then found that the mec1Δ, mre11Δ, and mec1Δ tel1Δ strains demonstrated increased rates of spontaneous loss of heterozygosity (LOH), which includes crossover, gene conversion, and chromosome loss, compared with the wild-type strain. In the tel1Δ strain, the rate of spontaneous LOH was as low as that in the wild-type strain. Finally, no induction of LOH by MMS was observed in the mec1Δ, mre11Δ, or mec1Δ tel1Δ strain; however, it was detected in the wild-type and tel1Δ strains upon exposure to MMS. The elevated level of spontaneous LOH but not MMS-induced LOH in the mec1Δ, mre11Δ, and mec1Δ tel1Δ strains suggests the presence of high levels of spontaneous recombinogenic DNA damage, which differs from the damage induced by MMS treatment, in these strains.
The intergenic region between the hemoglobin (hb) and nucleosome assembly protein-1 (nap-1) genes in the Paramecium caudatum macronuclear genome was previously found to be heterogeneously composed. Cloning of this intergenic region from the macronuclear genomic DNA identified four unique DNA fragments of different sizes. Sequencing of the cloned fragments revealed extreme heterogeneity and characteristics of both internal eliminated sequence (IES) and imprecise internal deletion sequences (IIDSs) in the intergenic region. Missing sequences were an AT-rich and direct repeats existed in their boundaries. Southern blotting of the total genomic DNA and polymerase chain reaction (PCR) of the total genomic DNAs indicated that there exist a dozen DNA fragments of different sizes in this intergenic region. It is likely that the heterogeneity found in the P. caudatum macronuclear genome results from the variable removal of an intergenic region.
We used two gametocidal (Gc) chromosomes 2C and 3CSAT to dissect barley chromosome 4H added to common wheat. The Gc chromosome induced chromosomal structural rearrangements in the progeny of the 4H addition line of common wheat carrying the monosomic Gc chromosome. We conducted in situ hybridization to select plants carrying rearranged 4H chromosomes and characterized the rearranged chromosomes by sequential C-banding and in situ hybridization. We established 60 dissection lines of common wheat carrying single rearranged 4H chromosomes. The rearranged 4H chromosomes had either a deletion or a translocation or a complicated structural change. The breakpoints were distributed in the short arm, centromere and the long arm at a rough ratio of 2:2:1. We conducted PCR analysis using the dissection lines and 93 EST markers specific to chromosome 4H. Based on the PCR result, we constructed a cytological map of chromosome 4H with 18 regions separated by the breakpoints of the rearranged chromosomes. Thirty-seven markers were present in the short arm and 56 in the long arm, and about 70% of the markers were present in no more than the distal 25.6% and 43.1% regions of the short and long arms, respectively. It is noteworthy that nine of the short-arm markers and 13 of the long-arm markers existed in the small subtelomeric regions at both ends characterized by the HvT01 sequences. We reconstructed a genetic map using 38 of the 93 markers that was used to construct the cytological map of chromosome 4H. The order of the markers on the genetic map was almost the same as that on the cytological map. On the genetic map, no markers were available in the pericentromeric region, but on the cytological map, 14 markers were present in the proximal region, and one of the markers was in the centromeric region of the short arm.
Mitochondrial functions are potential targets of abiotic stresses that are major environmental factors limiting plant development and productivity. To evaluate mitochondrial responses to abiotic stresses we studied mitochondrial transcriptome profiles at the early stages of wheat development after imbibition under normal and induced stress conditions. Three stresses given were low temperature (4°C), high salinity (0.2 M NaCl) and high osmotic potential (0.3 M mannitol). All these stresses greatly reduced growth but dramatically increased respiration both via the cytochrome and alternative pathways. Macroarray analysis of the mitochondrial transcriptome revealed that most of the changes in transcript levels were stress specific but groups of genes responded commonly to different stresses. Under 3-days continuous stresses, 13 genes showed low temperature specific responses with either up- or down-regulation, while 14 and 23 genes showed responses specific to high salinity and high osmotic potential, respectively. On the other hand, 13 genes showed common responses, among which cob and ccmFn increased their transcript levels while transcripts of the other genes including nad6, atp4 and atp9 decreased. The differential profiles of mitochondrial transcriptome revealed by the macroarray analysis were verified by the quantitative reverse transcriptase PCR analysis. Taken together, three among five nuclear-encoded mitochondria-targeted genes included in the array showed decreases under the stresses, while MnSOD and AOX increased their transcript amounts. Our results indicated the existence of common and different regulatory mechanisms that can sense different abiotic stresses and modulate both nuclear and mitochondrial gene expression in germinating wheat embryos and seedlings.
Several species of the genus Aegilops, wild relatives of wheat (Triticum aestivum, 2n = 6x = 42, AABBDD) carry gametocidal (Gc) genes. Gc genes kill the gametes without themselves by causing chromosomal breakage during post-meiotic cell divisions, and therefore are strong segregation distorters. The Gc gene Gc3-C1 derived from chromosome 3C of Ae. triuncialis (2n = 4x = 28, CCUU) induces chromosomal breakage in wheat cultivar ‘Chinese Spring’ (CS) but not in cultivar ‘Norin 26’ (N26). This cultivar-specific inhibition of Gc function is caused by a suppressor gene Igc1 located on chromosome 3B of N26. Igc1 is presumed to be a modified Gc gene without breakage function because of its homoeology to Gc3-C1. Here we report the results of linkage and physical mapping of Igc1 to help elucidate the molecular mechanisms underlying Gc action. Segregation analysis of the phenotypic data in BC1F1 mapping population of the cross between (CSxN26)F1 and CS + 3C” showed a 1:1 segregation ratio indicating that Igc1 is a dominant gene. In the linkage analysis, three molecular marker loci Xgwm285, Xgwm376, and Xcfp1886 cosegregated with the Igc1 locus. Bin mapping assigned the loci Xgwm285 and Xcfp1886 to bin C-3BS1-0.33 and Xgwm376 to bin C-3BL2-0.22. Physical mapping using Gc-induced chromosomal deletion lines of chromosome 3B of N26 revealed that the Igc1 locus resides in 52.0% or 2.1% of bins C-3BS1-0.33 and C-3BL2-0.22, respectively. Pericentromeric localization of Igc1 in chromosome 3B of N26 may have a positive effect to keep the two-component system of the Gc action. Map-based cloning approach to isolate the Igc1 may be difficult because recombination is depleted in the pericentromeric region. As is shown in this study, the combination of genetic and physical mapping offers high efficiency to identify the regions where genes are located especially in regions with low levels of recombination.
Genetic diversity of the wild population of the endangered Okinawa Rail, Gallirallus okinawae, was revealed by analyzing haplotypes in the mitochondrial control region for 177 individuals. We found 6 haplotypes with nucleotide differences at 6 sites. The four major haplotypes, Type 1 to Type 4, were present in 121 (68.4%), 21 (11.9%), 8 (4.5%) and 25 individuals (14.1%), respectively. Type 5 and Type 6 were each found in one individual. The gene diversity (h) and nucleotide diversity (π) of Okinawa Rail were calculated to be 0.499 ± 0.040 and 0.00146 ± 0.00098, respectively. Gene diversity in Okinawa Rail is higher than that found in other endangered avian species, but the relative nucleotide diversity is lower due to few nucleotide differences among the haplotypes. Our sample of 177 individuals represents 20–25% of the total population, and thus allows a rigorous estimate of the population structure of Okinawa Rail, and makes it unlikely that more haplotypes would be found with additional sampling. The low nucleotide diversity in the control region may indicate that Okinawa Rail has gone through a recent bottleneck. The minimal span network of haplotypes, and the distribution pattern of sampled individuals, indicate that the number of birds with rare haplotypes, Type 5 and 6, decreased during the recent population decline caused by habitat loss and introduced predators. Our results are relevant to the current conservation program for the endangered Okinawa Rail, and perhaps for other species of flightless rails.
The ADRA1A (Alpha-1-adrenergic receptor) gene is one of the members of G protein-coupled receptor superfamily. Alternative splicing of this gene was known to generate four transcript variants which code four isoforms with various C-terminal regions. In this study, we conducted expression analysis and evolutionary characterization of alternative transcripts of the ADRA1A gene. In total, 10 alternative transcripts were identified using experimental approaches and in silico analysis. Among them, 6 alternative transcripts (T1, T2, T3, T4, T4-1, and T8) were validated by RT-PCR approaches and sequencing procedures. From the alternative splicing analysis, it could be assumed that there were three different alternative transcripts generation mechanisms and unknown mechanism. First one is the integration event of three different TEs (AluSc, L1MC5, and MIR3) as seen on the last exons of T3, T4, T4-1, T5, T6, and T7 transcripts. The second mechanism is a differential promoter usage on T8. The third one is a substitution event of the 3’ splicing site during the primate evolution on T3. The last one is an unknown mechanism which was identified in the T4-1 transcript. Therefore, alternative transcripts of human ADRA1A gene occurred by four different ways, such as integration of TEs, differential promoter usage, substitution of splicing sites, and unknown mechanism.
GASDERMIN B (GSDMB) belongs to the novel gene family GASDERMIN (GSDM). All GSDM family members are located in amplicons, genomic regions often amplified during cancer development. Given that GSDMB is highly expressed in cancerous cells and the locus resides in an amplicon, GSDMB may be involved in cancer development and/or progression. However, only limited information is available on GSDMB expression in tissues, normal and cancerous, from cancer patients. Furthermore, the molecular mechanisms that regulate GSDMB expression in gastric tissues are poorly understood. We investigated the spatiotemporal expression patterns of GSDMB in gastric cancer patients and the 5’ regulatory sequences upstream of GSDMB. GSDMB was not expressed in the majority of normal gastric-tissue samples, and the expression level was very low in the few normal samples with GSDMB expression. Most pre-cancer samples showed moderate GSDMB expression, and most cancerous samples showed augmented GSDMB expression. Analysis of genome sequences revealed that an Alu element resides in the 5’ region upstream of GSDMB. Reporter assays using intact, deleted, and mutated Alu elements clearly showed that this Alu element positively regulates GSDMB expression and that a putative IKZF binding motif in this element is crucial to upregulate GSDMB expression.
The last sentence on p. 286 "In both R. apiculata and R. mucronata pollen release is very limited (Kusmana, personal communication), which could cause increased levels of inbreeding." should be deleted.