DNA polymerases synthesize new DNA strands according to the template DNA, using deoxynucleotide triphosphates during DNA replication and repair, and are essential to maintain genome integrity in DNA metabolism. In addition, these enzymes are widely used for genetic engineering techniques, including dideoxy-sequencing, PCR, DNA labeling, mutagenesis, and other in vitro experiments. Thermostable DNA polymerases are especially useful for PCR and cycle-sequencing. We propose a powerful strategy using environmental DNA as a genetic resource to investigate the structure-function relationships of the family B DNA polymerases. The region corresponding to the active center of the DNA polymerizing reaction in the structural gene of P. furiosus DNA polymerase I (PolBI) was substituted by PCR fragments amplified from DNAs within soil samples from various locations in Japan. The chimeric pol genes were constructed within the PolBI expression plasmid. The chimeric enzymes thus produced revealed DNA polymerase activities with different properties.
SsrA is a small RNA playing a crucial role in trans-translation, which leads to rescue of stalled ribosomes on or at the end of mRNA and addition of the degradation tag to a growing polypeptide. The lack of SsrA has been shown to enhance the temperature-sensitive (ts) phenotype of an E. coli strain defective in the degP gene, which encodes one of the periplasmic proteases. This severe ts phenotype was relieved only partially by an SsrADD variant, which can lead to ribosome rescue but adds a protease-resistant tag instead of the degradation tag, suggesting that accumulation of polypeptides programmed by truncated mRNAs is responsible for growth defect of the ssrA degP mutant. Expression of an S210A-mutant DegP protein, which lacks the protease activity but retains the chaperone activity, could relieve the ts phenotype of the double mutant, suggesting that the chaperone activity but not the protease activity of DegP is required for growth of the ssrA-deficient cells at high temperature. Overexpression of the rpoE gene, which encodes σE responsible for the expression of factors involved in extracellular stress response, also suppressed the ts phenotype of the ssrA degP mutant. This suggests that the stress-responsing pathway(s) may be involved in the enhancement of ts phenotype of degP mutant in the absence of SsrA.
We used the gametocidal system to dissect a barley chromosome 3H added to common wheat. The gametocidal system induced chromosomal structural changes in the 3H addition line of common wheat, and we cytologically screened for rearranged chromosomes involving the 3H chromosome by in situ hybridization (FISH/GISH). We established 50 common wheat lines carrying single rearranged (or dissected) 3H chromosomes of independent origin. The dissected 3H chromosomes were either deletions or translocations with wheat chromosomes, and their breakpoints were in the centromere/the long arm/the short arm in a rough ratio of 1:2:2. We used these so-called 3H dissection lines to map 36 EST markers that were polymorphic between euploid common wheat and the 3H addition line and that had been used for the construction of a 3H genetic map. We conducted PCR analysis to detect the EST markers in the dissection lines. The results of the PCR analysis, which mostly corresponded to the retained or lost segments of the dissected 3H chromosomes, allowed us to place the 36 EST markers into 20 chromosomal regions flanked by the breakpoints of the dissected chromosomes. We compared this physical map constructed in this study with a 3H genetic map constructed using the same EST markers. The order of all EST markers was consistent between the two maps. We briefly discuss on the advantage of the physical mapping using dissection lines over genetic mapping.
It has been shown that the disruption of the α-subunit gene of heterotorimeric G-proteins (Gα) results in dwarf traits, the erection of leaves and the setting of small seeds in rice. These mutants are called d1. We have studied the expression profiles of the transcripts and translation products of rice Gα in ten alleles of d1 including five additional alleles newly identified. By RT-PCR, the transcripts of the Gα gene were detected in the all d1 alleles. By western blot, the Gα proteins were not detected in the plasma membrane fractions of the d1 alleles with the exception of d1-4. In d1-4, one amino acid change in the GTP-binding box A of the Gα protein was occurred and even in this case the Gα protein was only just detectable in the plasma membrane fraction. Given that the Gα protein did not accumulate in the plasma membrane fraction in d1-8 which has a deletion of just a single amino acid in the Gα protein, it is likely that a proper conformation of the Gα is necessary for accumulation of Gα protein in the plasma membrane. Nine alleles of d1 showed a severer phenotype whilst d1-4 exhibited a mild phenotype with respect to seed size and elongation pattern of internodes. As brassinosteroid signaling was known to be partially impaired in d1s, the sensitivity to 24-epibrassinolide (24-epiBL) was compared among d1 alleles in a T65 genetic background. Only d1-4 showed responses similar to wild type rice. The results show that the d1-4 mutant is a mild allele in terms of the phenotype and mild hyposensitivity to the exogenously applied 24-epiBL.
To develop the bunching onion (Allium fistulosum L.; genomes, FF) chromosome-specific genetic markers for identifying extra chromosomes, eight shallot (A. cepa L. Aggregatum group; genomes, AA) – A. fistulosum monosomic addition plants (AA+nF) and 62 shallot – A. fistulosum single-alien deletion plants (AAF-nF) were analyzed by 23 different chromosome-specific genetic markers of shallot. The eight monosomic addition plants consisted of one AA+2F, two AA+6F, and five AA+8F. Of the 62 single-alien deletion plants, 60 could be identified as six different single-alien deletion lines (AAF-1F, -3F, -4F, -6F, -7F, and -8F) out of the eight possible types. Several single-alien deletion lines were classified on the basis of leaf and bulb characteristics. AAF-8F had the largest number of expanded leaves of five deletion plants. AAF-7F grew most vigorously, as expressed by its long leaf blade and biggest bulb size. AAF-4F had very small bulbs. AAF-7F and AAF-8F had different bulbs from those of shallot as well as other types of single-alien deletion lines in skin and outer scale color. Regarding the sugar content of the bulb tissues, the single-alien deletion lines showed higher fructan content than shallot. Moreover, shallot could not produce fructan with degree of polymerization (DP) 12 or higher, although the single-alien deletion lines showed DP 20 or higher. The content of S-alk(en)yl-L-cysteine sulfoxide (ACSO) in the single-alien deletion lines was significantly lower than that in shallot. These results indicated that chromosomes from A. fistulosum might carry anonymous factors to increase the highly polymerized fructan production and inhibit the synthesis of ACSO in shallot bulbs. Accordingly, alien chromosomes from A. fistulosum in shallot would contribute to modify the quality of shallot bulbs.
In some arthropods, paternal chromosomes are inactivated or eliminated in adult males and are not transmitted to offspring by sperm. This unique chromosome system is called paternal genome loss (PGL). In scale insects (Hemiptera; superfamily Coccoidea), PGL is widespread and three types of PGL have been identified. The questions as to whether PGL is of single origin or of multiple origins, and whether PGL is evolved to haplodiploidy or derived from diplodiploidy such as XX-XO have remained areas of controversy. Preliminary phylogenetic analyses using nuclear ribosomal DNA of 495 nucleotide sites failed to provide conclusive answers to these questions. Here we report a highly-resolved phylogeny of scale insects based on 1,229 nucleotide sites from the mitochondrial genes (COI and COII). The paraphyly of Archaeococcoidea and the monophyly of Neococcoidea are strongly supported by Bayesian posterior probabilities p = 0.99 and 1.00, respectively. These two hypotheses are supported also by maximum likelihood bootstrap probabilities BP = 79.9 and 99.4%, respectively. The relationships among families of Neococcoidea are resolved, being supported by p = 0.90–1.00 and BP = 58.7–100%. Thus, the phylogenetic tree provides us a sound basis for reconstructing the evolutionary history of PGL in scale insects. Such results have demonstrated that (1) the common ancestor of scale insects was diplodiploidy of the XX-XO sex determination, (2) PGL has a single origin from XX-XO in the common ancestor of Neococcoidea, and (3) haplodiploidy was derived from XX-XO, but not from PGL. These results support the theories arguing that PGL is an evolutionary stable state.
Co-inheritance of α-thalassemia with homozygosity or compound heterozygosity for β-thalassemia may ameliorate β-thalassemia major. A wide range of clinical phenotypes is produced depending on the number of α-thalassemia alleles (-α/αα --/αα, --/-α). The co-inheritance of β-thalassemia with α-thalassemia with a single gene deletion (-α/αα) is usually associated with thalassemia major. In contrast, the co-inheritance of β-thalassemia with two α-genes deleted in cis or trans (--/αα or -α/-α) generally produces β-thalassemia intermedia. In Southeast Asia, the most common defect responsible for α-thalassemia is the Southeast Asian (SEA) deletion of 20.5 kilobases. The presence of the SEA deletion with Hb Constant Spring (HbCS) produces HbH-CS disease. Co-inheritance of HbH-CS with compound heterozygosity for β-thalassemia is very rare. This study presents a Malay patient with HbH-CS disorder and β°/β+-thalassemia. The SEA deletion was confirmed in the patient using a duplex-PCR. A Combine-Amplification Refractory Mutation System (C-ARMS) technique to simultaneously detect HbCS and Hb Quong Sze confirmed HbCS in the patient. Compound heterozygosity for CD41/42 and Poly A was confirmed using the ARMS. This is a unique case as the SEA α-gene deletion in cis (--SEA/αα) is generally not present in the Malays, who more commonly posses the two α-gene deletion in trans (-α/-α). In addition, the β-globin gene mutation at CD41/42 is a common mutation in the Chinese and not in the Malays. The presence of both the SEA deletion and CD41/42 in the mother of the patient suggests the possible introduction of these two defects into the family by marriage with a Chinese.
Ortho-phenyl phenol and its hepatic derivative, phenyl hydroquinone, do not generate base-substitution-type mutations, but cause bladder cancer in rats and mice. The mechanism of their carcinogenic effect is unknown. We have previously shown that o-phenyl phenol and phenyl hydroquinone induce mitotic arrest and aneuploidy in Saccharomyces cerevisiae. To further delineate the mechanism of action of phenyl hydroquinone, we examined its effect on human cells. Treatment of the colon cancer cell line HCT116 with 0 to 150 μM phenyl hydroquinone caused a concentration-dependent inhibition of growth, accumulation of cells having G2/M DNA content, and an increase in the mitotic index. Moreover, a dose-dependent increase in apoptotic cells was observed. Finally, a high frequency of aneuploid cells was found. On the other hand, no increase in γ-H2AX foci was observed. The results show that phenyl hydroquinone does induce mitotic arrest, apoptosis and aneuploidy in the absence of DNA damage. Our results may be useful to understand the mechanisms of action of chemical substances that are Ames test-negative carcinogens.
In this paper a novel genetic algorithm is presented for the dyad motif finding problem. The genetic algorithm uses a multi-objective fitness function based on the sum of pairs, the number of matches, and the information content. The individuals required for the population pool in the genetic algorithm are optimized by Gibbs sampling method. Also, new crossover and mutation operators are designed. The algorithm is implemented and tested on the different types of real datasets. The results are compared with other well-known algorithms and the effectiveness of our algorithm is shown.
Transcription factors, RAV1 and RAV2 from Arabidopsis thaliana, contain two distinct DNA-binding domains, AP2/EREBP and B3, both of which are uniquely found in plants. We found that transcripts of RAV1 and RAV2 were upregulated transiently by touch-related mechanical stimuli. However, the temporal expression patterns of RAV1 and RAV2 differed from those of known touch-induced genes. A striking feature of mechanical stimulus-induced expression of RAV1 and RAV2 was that it was biphasic; the RAV1 and RAV2 expression was reinduced and sustained after a rapid and transient induction. The extent of both transient and subsequent upregulation by touch-stimuli depended on the dose of the initial stimulus. Analysis of transgenic A. thaliana plants carrying a RAV2 promoter-GUS fusion gene indicated that the transient mechanical stimulus-induced RAV2 expression was primarily controlled by its promoter. Histochemical analysis of the transgenic plants revealed that GUS expression was strongly induced in the petioles and primordia of true leaves and shoot apical meristems, which may be related to the alteration in plant growth pattern caused by touch-stimuli. Because RAV1 has been suggested to be a negative regulator of growth and development, the dose-dependent biphasic upregulation of RAV1 and RAV2 may serve not only for immediate physiological responses and but also for developmental adaptation in response to the environmental stimuli.