When the dmd gene of bacteriophage T4 is defective, expression of middle genes starts normally but drops abruptly. However, the residual expression of middle genes at late stages continues at a higher rate in cells infected with a dmd mutant than with the wild type. In order to understand the complex effects of the dmd gene, we followed changes in the quantity of mRNA from a middle gene, uvsY. The uvsY mRNA was degraded rapidly by RNase LS at middle stages but stabilized at late stages, suggesting that RNase LS targets middle-gene mRNAs only at middle stages. Furthermore, another RNase targeting middle mRNAs at late stages is also suggested to be inactivated when dmd is mutated. We found that RNase E was involved in the degradation of uvsY mRNA. Judging from the processing of gene-32 mRNA, RNase E activity declines after the beginning of the middle stage when dmd is defective.
We used the intergenic spacer sequences of the 5S ribosomal RNA genes (5S rDNA) to obtain insights into the genomic origin of putative amphidiploid/tetraploid species with 2n = 48 and their descendants in Nicotiana. Amplification of the spacer sequences and subsequent multiple alignment using the consensus sequences from each species, showed that two Australian species shared common large deletions, suggesting that the origin of the 5S rDNA is closely related in these species. Comparison of the spacer sequences with those from diploid (2n = 24) Nicotiana species made it possible to detect some groups consisting of the sequences from the 2n = 24 and 2n = 48 level species. Chromosomal localizations of the 5S rDNA arrays were similar in most groups. The relationships suggested by the 5S rDNA were also assessed at the genome level by using genomic in situ hybridization. We showed that the grouping based on the 5S rDNA spacer sequence reflects high genomic homology between 2n = 24 and 2n = 48 level species. As a result, the putative polyploid species such as N. debneyi, N. quadrivalvis, and N.africana were suggested to involve the close relatives of the diploid species such as N. glauca, N.obtusifolia and N. sylvestris, and N. langsdorffii, respectively, in their speciation. Our results are generally in agreement with the relationships previously suggested by morphological and cytogenetic observations, and some novel relationships were also revealed.
Transacting factors often form homo- and heterodimers and regulate various targets, the type of regulation depending on the dimeric combination. The WUS and TALE subfamilies are two atypical homeodomains in plants. A homeodomain mediates sequence-specific binding to its target DNA and usually consists of 60 amino acid residues, whereas atypical homeodomains have extra amino acid residues in the well-conserved region. The genes OsWUS and OsPRS, which encode atypical homeodomain proteins from the WUS subfamily, and OsBEL and OSH15, which encode those from the TALE subfamily, were isolated from rice and tested for their interactions by yeast two-hybrid analysis. OsWUS and OsPRS formed homodimers and formed heterodimers with each other but did not form dimers with the TALE family homeodomain proteins OSH15 or OsBEL. Likewise, OSH15 and OsBEL formed homodimers and heterodimers but did not form dimers with the WUS family homeodomain proteins OsWUS and OsPRS. These findings suggest that the combinations of dimers are well correlated with the classification of these proteins on the basis of sequence similarity. RT-PCR analysis revealed that expression of OsWUS and OsPRS was detected in the same organs, namely floral buds, roots, and suspension cells. Therefore, it is possible that the proteins encoded by both of these genes function as homo- and heterodimers in planta. These results suggest that, during the evolution of these subfamilies, various combinations of dimers within proteins encoded by paralogous genes were formed and generated independent regulatory networks that enabled complex patterns of plant development.
Nucleosomal histones are covalently modified at specific amino acid residues. In the case of histone H4, four lysines (K5, K8, K12, and K16) are acetylated. In the current studies, we examined the dynamics of histone H4 acetylation at K8 and K12 in mitotic barley cells using a three-dimensional immunofluorescent method. Based on the results and previous studies on the dynamics of K5 and K16 acetylation, we provide a comprehensive view of the dynamics of H4 acetylation. Interphase nuclei exhibit strong acetylation in the centromeric region at K5, K8 and K12. In the case of K12, strong acetylation at nucleolar organizing regions was observed from prophase to anaphase. The dynamics of K12 were closely related to those of K5. On the other hand, K8 exhibited a pattern of almost uniform acetylation from prophase to telophase and strong acetylation in distal regions of chromosomes at both metaphase and anaphase, which is very similar to the dynamics of K16 acetylation. Thus, it appears that there is pair-wise acetylation of K12 and K5 in the nucleolar organizing regions and of K8 and K16 in the gene-rich regions. Together, these results suggest that pair-wise dynamics of H4 acetylation regulate chromosomal structure and function during the cell cycle.
The second largest BamHI fragment (B2) of the chloroplast DNA in Triticum (wheat) and Aegilops contains a highly variable region (a hotspot), resulting in four types of B2 of different size, i.e. B2l (10.5kb), B2m (10.2kb), B2 (9.6kb) and B2s (9.4kb). In order to gain a better understanding of the molecular nature of the variations in length and explain unexpected identity among B2 of Ae. ovata, Ae. speltoides and common wheat (T. aestivum), the nucleotide sequence between a stop codon of rbcL and a HindIII site in cemA in the hotspot was determined for Ae. ovata, Ae. speltoides, Ae. caudata and Ae. mutica. The total number of nucleotides in the region was 2808, 2810, 3302, and 3594 bp, for Ae. speltoides, Ae. ovata, Ae. caudata and Ae. mutica, respectively, and the sequences were compared with the corresponding ones of Ae. crassa 4x, T. aestivum and Ae. squarrosa. Compared with the largest B2l fragment of Ae. mutica, a 791bp and a 793 bp deletion were found in Ae. speltoides and Ae. ovata, respectively, and the possible site of deletion in the two species is the same as that of T. aestivum. However, a deleted segment in Ae. ovata is 2 bp longer than that of Ae. speltoides (and T. aestivum), demonstrating that recurrent deletions had occurred in the chloroplast genomes of both species. Comparison of the sequences from Ae. caudata and Ae. crassa 4x with that of Ae. mutica revealed a 289 bp and a 61 bp deletion at the same site in Ae. caudata and Ae. crassa 4x, respectively. Sequence comparison using wild Aegilops plants showed that the large length variations in a hotspot are fixed to each species. A considerable number of polymorphisms are observed in a loop in the 3’ of rbcL. The study reveals the relative importance of the large and small indels and minute inversions to account for variations in the chloroplast genomes among closely related species.
Under overdominant selection, mutants substantially contribute to increase the amount of polymorphism. It is also known that under neutrality as the migration rates among demes decrease in a subdivided population, the amount of polymorphism increases along with the increase of the effective population size, Ne. In this study, under overdominant selection the effect of population subdivision on the amount of polymorphism was investigated using the diffusion approximation and the low migration approximation. It was shown that if selection is medium or strong (e.g., NTs > 1, where NT is the population size and s is the selective advantage of heterozygotes), the nucleotide diversity, π, decreases along with the decrease of Nm against the increase of Ne, where N is the size of demes and m is the migration rate per deme. In addition, the ratio of the nucleotide diversity to the evolutionary rate also decreases along with the decrease of Nm. In some cases the ratio becomes smaller than that expected under neutrality as Nm decreases.
Heteroplasmic nucleotide polymorphisms are rarely observed in wild animal mitochondrial DNA. The occurrence of such site heteroplasmy is expected to be extremely rare at nonsynonymous sites where the number of nucleotide substitutions per site is low due to functional constraints. This report deals with nonsynonymous mitochondrial heteroplasmy from two wild fish species, chum salmon and Japanese flounder. We detected an A/C nonsynonymous heteroplasmic site corresponding to putative amino acids, Ile or Met, in NADH dehydrogenase subunit-5 (ND5) region of chum salmon. The heteroplasmic site was at the 3rd position of 58th codon. As for Japanese flounder we detected a C/T nonsynonymous heteroplasmic site corresponding to putative amino acids, Leu or Pro, in ND4 region. The heteroplasmic site was at the 2nd position of 450th codon. We also verified heteroplasmy at these sites by sequencing cloned fragments.
Polymorphisms in the prion protein gene (PRNP) in humans and sheep correlate with susceptibility to transmissible spongiform encephalopathies (TSEs). Bovine spongiform encephalopathy (BSE) has been reported in British and Japanese cattle; it has occurred thus far in Holstein cattle. BSE in Hanwoo (Bos taurus coreanae) cattle has not been diagnosed up to now. To characterize the bovine PRNP polymorphisms in Korean cattle, we analyzed the open reading frame (ORF) of PRNP in 120 Hanwoo (beef) cattle and 53 Holstein (dairy) cattle. Three polymorphisms were found, the third position of codon 78 (G→A), the third position of codon 192 (C→T), and the deletion of a single octa-repeat. An analysis of codon 78 revealed no difference in the genotype (P = 0.2026) or allele (P = 0.7180) frequencies between Hanwoo and Holstein animals. However, there were significant differences in the genotype (P < 0.0001) and allele (P < 0.0001) frequencies at PRNP codon 192 between Hanwoo and Holstein animals. The rate of Holstein animals with deletion of a single octa-repeat was 91.5% undeleted homozygotes, 8.5% heterozygotes (with R3 deletion), and 0% deleted homozygotes. However, none of the 120 Hanwoo animals had any octa-repeat deletions. The genotype (P < 0.0001) and allele (P < 0.0001) frequencies of a single octa-repeat-deletion were also significantly different between Hanwoo and Holstein animals.