In the nuclei of some interspecific hybrid and allopolyploid plant species, each genome occupies a separate spatial domain. To analyze this phenomenon, we studied localization of the centromeres in the nuclei of a hybrid between Torenia fournieri and T. baillonii during mitosis and meiosis using three-dimensional fluorescence in situ hybridization (3D-FISH) probed with species-specific centromere repeats. Centromeres of each genome were located separately in undifferentiated cells but not differentiated cells, suggesting that cell division might be the possible force causing centromere separation. However, no remarkable difference of dividing distance was detected between chromatids with different centromeres in anaphase and telophase, indicating that tension of the spindle fiber attached to each chromatid is not the cause of centromere separation in Torenia. In differentiated cells, centromeres in both genomes were not often observed for the expected chromosome number, indicating centromere association. In addition, association of centromeres from the same genome was observed at a higher frequency than between different genomes. This finding suggests that centromeres within one genome are spatially separated from those within the other. This close position may increase possibility of association between centromeres of the same genome. In meiotic prophase, all centromeres irrespective of the genome were associated in a certain portion of the nucleus. Since centromere association in the interspecific hybrid and amphiploid was tighter than that in the diploid parents, it is possible that this phenomenon may be involved in sorting and pairing of homologous chromosomes.
The DNA polymorphism in the AtPrx53 gene which encodes anionic peroxidase was analyzed in 20 Arabidopsis thaliana accessions. There are two divergent sequence types (Col and Dj-like haplotypes) in the AtPrx53 gene that differ by 2 indel and 16 non-singleton nucleotide polymorphisms including 5 nucleotide polymorphic sites responsible for 4 deduced amino acid replacements. Two of the amino acid substitutions (Phe/Ser180and Asp/Asn270) could be responsible for the difference in electrophoretic mobility of AtPrx53 allozymes. One of them (Phe/Ser180) lies within the hypervariable region, indicating that this amino acid polymorphism is subjected to balancing selection. The revealed difference between deduced allozymes is related to the dimorphism in mobility of three major anionic peroxidase isoforms which according to previously established data encoded by AtPrx53 gene. The haplotype Col which included 12 accessions from three different continents is characterized by faster mobility of three isoforms in comparison with the Dj haplotype represented by eight accessions. There is a significant association between the haplotype and several developmental traits: leaf number, flowering time, main stem height etc. Lines of the Dj haplotype have shorter duration of vegetative stages and flower earlier than most of Col haplotype accessions. The reasons of this association are discussed.
Maize MuDR/Mu elements are one of the most active Class II transposons and are widely used for transposon tagging for gene cloning. The autonomous MuDR encodes a transposase, while diverse non-autonomous elements share similarity to MuDR only within their ~215 bp terminal inverted repeats (TIRs). Four independent Mu-induced mutable alleles of the anthocyanin pigment pathway Bronze2 (Bz2) locus have been sequenced; bz2-mu1, bz2-mu2, and bz2-mu3 contain Mu1 element insertions while bz2-mu4 contains a MuDR insertion. Somatic excision activity can be monitored for each allele as a purple spotted phenotype on the otherwise beige epidermal layer of the kernel. To study epigenetic silencing of Mu elements, we investigated inheritance of somatic transposition of these four reporter alleles, and using samples from leaves just below the ear and the tassel, DNA methylation status and mudrA expression were quantified through three consecutive generations. Percentages of spotted kernels when crossed into bz2 tester as pollen parent were lower than those when crossed with bz2 tester as ear parent in all bz2-mu alleles. The propensity for silencing in kernels with different frequencies of spotting was investigated. In the inactive lines, both MuDR and Mu1 elements were denovo methylated. The transposition frequency was negatively correlated with the level of Mu methylation and positively correlated with the level of mudrA transcript. The most reliable indicator of incipient silencing was a decrease in mudrA transcript levels in the leaf below the tassel, and this transcriptional silencing could precede methylation of Mu elements.
Alien chromosome addition lines are useful genetic material for studying the effect of an individual chromosome in the same genetic background. However, addition lines are sometimes unstable and tend to lose the alien chromosome in subsequent generations. In this study, we report preferential removal of chromosome 1D rather than the alien chromosome from homoeologous group-1 addition lines. The Agropyron intermedium chromosome 1Agi (1E) addition line, created in the background of ‘Vilmorin 27’, showed loss of a part of chromosome 1D, thereby losing its HMW glutenin locus. Even in the case of Aegilops longissima and Ae. peregrina, the genomes of which are closer to the B genome than D genome, chromosome 1D was lost from chromosome 1Sl and 1Sv addition lines in cv. ‘Chinese Spring’ rather than chromosome 1B during transfer from one generation to another. A similar observation was also observed in the case of a chromosome 1E disomic addition line of Ag. elongatum and alloplasmic common wheat line with Ag. intermedium ssp. trichophorum cytoplasm. The reason for this strange observation is thought to lie in the history of wheat evolution, the size of chromosome 1D compared to 1A and 1B, or differing pollen competition abilities.
Germination of imbibed embryos is the initial stage of plant development that is accompanied by the burst of mitochondrial respiration. To understand the process of mitochondrial biogenesis during this critical stage in wheat development, we monitored changes in mitochondrial transcript profiles during the first 3 days by adopting a newly devised macroarray system. The whole experiment was conducted in the dark to avoid influences of photosynthesis. Dry quiescent embryos started respiration rapidly after imbibition and the rate of oxygen uptake increased to peak at the first day followed by a continuous decrease until the third day under this condition. Both the cytochrome and alternative electron transport pathways appeared to contribute to this initial burst. Shoot and root growth was also remarkable during this period. Mitochondrial transcriptome was studied by macroarray analysis using 28 mitochondrial protein-coding genes, 4 nuclear encoded mitochondria-targeted genes and 2 nuclear genes as control. All transcripts were present in dry embryos at different initial levels, and a large variability was observed in their abundance among individual genes throughout the tested period. Gene expression was categorized into four clusters according to the profiles of individual transcript accumulation. A majority of the genes encoding subunits of the respiratory complexes belonged to two major clusters, the time course of transcript accumulation of one cluster agreeing with that of respiratory development and the other remaining at high constant levels. The macroarray system devised in this study should be useful in monitoring mitochondrial biogenesis under various growth conditions and at different developmental stages in cereals.
House mouse (Mus musculus) is one of the perilous animal vectors for imported zoonosis such as a lymphocytic choriomeningitis (LCMV) infectious disease, and probably unknown emerging and/or re-emerging infectious diseases as well. It is necessary to prevent such diseases by regular surveys for behavioral trends of these allochthonous mice. However, such a trial has never been attempted in Japan. From 1998 to 2002, we analyzed partial sequences of the D-loop region in mtDNA, which provides powerful diagnostic SNPs for subspecies identification in the Mus musculus species, from 301 individuals of mice collected in 23 international bays or airports in Japan. We found that invasion of many allochthonous mice, which were identified as European subspecies, Mus musculus domesticus, occurred in Tokyo metropolitan coastal area. Based on the evidence, we warn that extensive invasion of allochthonous mice has occurred recently and, therefore, the risk of emerging and/or re-emerging infectious diseases invasion might be high in Tokyo metropolitan area.
A method of simultaneous isolation of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) from human blood has been proposed by improvising Lahiri’s method of isolation of nuclear DNA. The approach presented here provides selectively enriched fractions and eliminates the need for two different methods or separate reagent sets for the extraction of nDNA and mtDNA. It employs an initial nuclear/ cytoplasm partitioning, followed by the similar procedural steps for the two fractions separately. It gives good quality and quantity of the nDNA as well as the mtDNA, suitable for processes like PCR amplification and sequencing and may prove to be useful for people studying population genetics and evolution using molecular markers maximizing the available resources, especially in cases where a large database needs to be generated from limited amount of blood sample. From 3 ml of blood, the yields of mtDNA salvaged from the supernatant were sufficient to set approximately 4 × 105 reactions (starting with 250 fg DNA per reactions) of mtDNA loci which otherwise would have been discarded as per original Lahiri’s procedure. The quality of mtDNA from the mitochondrial fraction was suitable for all major downstream processes as confirmed by locus specific PCR amplifications and sequencing. Through this procedure, the wastage of nDNA can be avoided when mtDNA loci is studied.
The DNA binding protein H-NS promotes homologous recombination in Escherichia coli, but the role of its paralog StpA in this process remains unclear. Here we show that an hns mutant, but not an stpA mutant, are marginally defective in conjugational recombination and is sensitive to the double-strand-break-inducing agent bleomycin. Interestingly, the hns stpA double mutant is severely defective in homologous recombination and more bleomycin-sensitive than is the hns or stpA single mutant, indicating that the stpA mutation synergistically enhances the defects of homologous recombination and the increased bleomycin-sensitivity in the hns mutant. In addition, the transduction analysis in the hns stpA double mutant indicated that the stpA mutation also enhances the defect of recombination in the hns mutant. These results suggest that H-NS plays an important role in both homologous recombination and repair of bleomycin-induced damage, while StpA can substitute the H-NS function. The recombination analysis of hns single, stpA single, and hns stpA double mutants in the recBC sbcA and recBC sbcBC backgrounds suggested that the reduction of the hns single or hns stpA double mutants may not be due to the defect in a particular recombination pathway, but may be due to the defect in a common process of the pathways. The model for the functions of H-NS and StpA in homologous recombination and double-strand break repair is discussed.
The building of genetic maps in diploid organisms by crosses between different genotypes and estimation of recombination frequencies from the obtained segregation data has been successfully used since a very young step in the birth of genetics. The three-point cross methodology has facilitated this task and has demonstrated at the same time that genetic distances are not additive, as some recombinant products are not recognised in the progeny. Three-point cross also allows to examine if chiasma interference exists and its evaluation. Here I show that the classical method of this estimation is erroneous and inevitably determines the apparition of a spurious, positive interference, which has been claimed to be an almost general phenomenon. Interference can only be estimated from a precise knowledge of the number of crossing over events occurring in meiocytes.