Meiotic recombination is initiated by the introduction of DNA double-strand breaks (DSBs) at recombination hotspots. DSB ends are resected to yield ssDNA, which is used in a homology search. Sae2p, which is involved in the resection of DSB ends, is phosphorylated by the Mec1p and Tel1p kinases during meiosis. To clarify the role of Sae2p phosphorylation in meiotic recombination, three mutants with alanine substitutions (at two putative Mec1/Tel1 phosphorylation sites near the N terminus, at three sites near the C terminus or at all five sites) were constructed. Analysis of DSB ends during meiotic recombination demonstrated that phosphorylation of the three C-terminal phosphorylation sites is necessary for DSB end resection and that phosphorylation of the two N-terminal phosphorylation sites is required for the efficient initiation of DSB end resection. Sae2p was localized on meiotic chromosomes in the rad50S and mre11-H125R mutants, which accumulate DSB ends. Alanine substitutions of all phosphorylation sites did not affect localization of Sae2p on meiotic chromosomes. Although colocalization of Sae2p with Mre11p and recombinant formation were observed in the N-terminally mutated and the C-terminally mutated strains, these processes were drastically impaired in the quintuple mutant. These results indicate that phosphorylation of Sae2p is required to initiate resection and to improve the efficiency of resection through cooperation with the Mre11-Rad50-Xrs2 complex.
A predicted tau Glutathione S-transferase (GST) subunit encoding gene, named GhGST, was isolated from Gossypium hirsutum with RACE method from SSH library based on Verticillium dahliae stress. The data revealed an open reading frame of 678 bp encoding a protein of 225 amino acids with a molecular weight of 25.821 kDa. Semi-quantitative RT-PCR analysis showed that the mRNA of GhGST was expressed in root, stem and leaf. And the content of GhGST expression increased under Verticillium dahliae stress in root. The expression of GhGST gene was verified by transformation in E. coli BL21 (DE3) strain with the recombinant expression vector pET-32A. GST activity assay showed the crude GhGST protein had obvious activity to 1-chloro-2,4-dinitrobenzene (CDNB) substrate.
A database search of the sequences flanking a member of rice retrotransposon RIRE7 revealed that a 298-bp sequence in the region downstream of the member is a repetitive sequence interspersed in the genome of Oryza sativa cv. Nipponbare. Most of the repetitive sequences were flanked by a direct repeat of a target-site sequence, about 14 bp in length. The consensus sequence, 293 bp in length, had no regions encoding any proteins but had sequence motifs of an internal promoter of RNA polymerase III. These indicate that the sequence is a retroposon SINE, designated OsSN1 (Oryza sativaSINE1). OsSN1 is a new rice SINE, because it has no homology with any of the three p-SINE families previously identified from rice, and because it has a stretch of A at the 3’ end, unlike p-SINE and any other Gramineae SINEs which have a stretch of T at the 3’ end. The Nipponbare genome was found to have many members related to OsSN1, forming two additional new SINE families (designated OsSN2 and OsSN3). OsSN2 and OsSN3 are highly homologous to the 3’ and 5’ regions of OsSN1, respectively. This suggests that OsSN1 has a mosaic structure, which is generated by sequence exchange (or shuffling) between ancestral OsSN2 and OsSN3. Despite the absence of homology in the 3’ regions between OsSN1 (or OsSN2) and OsSN3, a sequence, 5’-TTCTC-3’, is commonly present in the region preceding the A stretch at the 3’ end. This sequence together with the A stretch and a stem-loop structure found in the region near the A stretch are assumed to be important for retroposition. OsSN members were present in strains of Oryza species, as were p-SINE members. Some of the members showed insertion polymorphism at the respective loci among the rice strains. p-SINE had such polymorphic members, which are useful for classification and phylogenetic analysis of various strains of Oryza species. The polymorphic members of OsSN were more frequently found than those of p-SINE, and therefore, such members are likely to be useful for extensive taxonomic and phylogenetic studies on various rice strains.
The DROOPING LEAF (DL) gene has an important function in rice development. Loss-of-function mutation of DL fails to form midrib, a strong structure formed in the central region of the leaf, resulting in a drooping leaf phenotype. In addition, severe mutation in DL causes a floral homeotic change, i.e., replacement of the carpels by the stamens. Thus, DL regulates midrib formation in the leaf and carpel specification in the flower. DL encodes a putative transcription factor that belongs to the YABBY family, suggesting that it plays an essential role in these developmental processes by regulating many downstream genes, either directly or indirectly. As a first step to elucidate genetic networks involved in midrib formation, in this paper, we attempt to identify genes regulated by DL in the initial processes of midrib development. By comparing expression profiles in the shoot apex that involves the leaf primordia, we found 90 genes that are upregulated at least two-fold higher in the transgenic plants overexpressing DL (Act1:DL) than in the null dl mutant (dl-sup1), suggesting that these genes are involved in midrib formation under the control of DL. Subsequent RT-PCR analysis of arbitrarily chosen 18 genes suggests that some genes may be involved in only midrib formation, whereas others may be involved in both midrib formation and carpel development.
We constructed and characterized arrayed bacterial artificial chromosome (BAC) libraries of five Drosophila species (D. melanogaster, D. simulans, D. sechellia, D. auraria, and D. ananassae), which are genetically well characterized in the studies of meiosis, evolution, population genetics, and developmental biology. The BAC libraries comprise 8,000 to 12,500 clones for each species, estimated to cover the most of the genomes. We sequenced both ends of most of these BAC clones with a success rate of 91%. Of these, 53,701 clones consisting of non-repetitive BAC end sequences (BESs) were mapped with reference of the public D. melanogaster genome sequences. The BES mapping estimated that the BAC libraries of D. auraria and D. ananassae covered 47% and 57% of the D. melanogaster genome, respectively, and those of D. melanogaster, D. sechellia, and D. simulans covered 94–97%. The low coverage by BESs of D. auraria and D. ananassae may be due to the high sequence divergence with D. melanogaster. From the comparative BES mapping, 111 possible breakpoints of chromosomal rearrangements were identified in these four species. The breakpoints of the major chromosome rearrangement between D. simulans and D. melanogaster on the third chromosome were determined within 20 kb in 84E and 30 kb in 93E/F. Corresponding breakpoints were also identified in D. sechellia. The BAC clones described here will be an important addition to the Drosophila genomic resources.
The odorant-binding protein (OBP) functions in chemosensation in insects. Two OBP genes, Obp57d and Obp57e, are involved in the evolution of the unique host-plant preference in Drosophila sechellia. Comparative analysis of the Obp57d/e genomic sequences in the Drosophila melanogaster species group has revealed that the rapid evolution of Obp57d and Obp57e has resulted in functional divergence between the two genes. Here, using D. melanogaster knockout strains generated by gene targeting, we examined the roles of Obp57d and Obp57e in behavioral response to a series of fatty acids. In the taste-based oviposition-site preference assay, the knockout flies showed stronger preference for acids than wild-type flies, indicating that the normal functions of Obp57d and Obp57e are associated with the suppression of positive preference for C6-C9 acids. Heterozygotes for each knockout haplotype also exhibited a significantly different behavioral response compared with wild-type flies, suggesting that Obp57d and Obp57e have a gene dosage effect on behavior. In contrast, the wild-type and knockout flies exhibited similar responses in the feeding assay and the odor-based free-walking assay, suggesting that the two OBPs’ contribution to feeding and olfactory behaviors is small. Taken together, our results demonstrated that each of Obp57d and Obp57e in D. melanogaster contributes to the determination of reproductive sites, suggesting that the two OBP genes play an important role in ecological adaptation of Drosophila.
Owing to the relevance to evolutionary theories of genotypic and phenotypic evolution, the correspondence of differentiation among natural populations in complex phenotypic traits and genetic markers has been studied extensively, and generally found to be poor. In contrast, the correspondence of differentiation among natural populations in gene expression, now often considered a genomic era proxy for the phenotype, and genetic markers, remains largely unexplored. Here, an analysis of expression and nucleotide sequence polymorphism of 106 genes in Drosophilamelanogaster strains of the Cosmopolitan (M) and Zimbabwe, Africa (Z) mating races showed that differentiation of gene expression and of coding sequences, measured as QST and GST, respectively, were uncorrelated and, generally, QST > GST. However, an exploratory analysis showed that GST of the 5 prime sequences of genes was correlated with QST calculated from expression data, while GST of the coding sequences remained uncorrelated with QST. This scenario is consistent with the population differentiation at cis-regulatory regions that is decoupled from differentiation of the coding regions. However, despite evidence for selection on global levels of gene expression (deduced from QST > GST), 5 prime sequence polymorphisms generally were compatible with selective neutrality, suggesting differentiation in cis-regulated gene expression for these genes has been promoted by drift or selection too weak or too long ago to be detected, or higher organizational levels underlying the genetic architecture of expression are targets of selection. In all, this raises the question how selection on the expression changes (i.e. the phenotype) can be so obvious yet elusive at the level of the nucleotide sequence. Our contrasts between genetic differentiation of populations in expression and sequences revealed that even when genotype and phenotype can be connected the sources of variation that are the target of selection remain to be identified.
The mitochondrial genomes of the Komodo monitor (Varanus komodoensis) and the Nile monitor (V. niloticus) were previously shown to have an extensive gene rearrangement. Here, we show that this gene arrangement widely occurs in varanid taxa originated from Africa, Asia and Australasia. Based on phylogenetic relationships of the varanids constructed using mitochondrial DNA sequences encoding the NADH dehydrogenase subunit 2 gene and seven flanking tRNA genes, we estimated their divergence times by the Bayesian method without assuming the molecular clock. The results suggested that the mitochondrial DNA gene rearrangement took place once in an ancestral varanid lineage in the Paleocene or earlier. Our results are more consistent with Cenozoic over-water dispersal of Southeast Asian varanids across the Indonesian Archipelago rather than the Cretaceous Gondwanan vicariance for the origin of Australasian varanids.
Small RNAs including microRNA (miRNA) and small interfering RNA (siRNA) are known as repressors of gene expression. There are many plant proteins involved in small RNA-mediated gene silencing, such as Dicer ribonucleases and RNA-dependent RNA polymerases. However, most of these proteins have been reported to be absent in the late developmental stage of the plant male gamete, pollen. In order to clarify the existence of the small RNAs during maturation of pollen, we cloned and sequenced small RNAs from rice anthers including tricellular pollen. From fifty six candidates of small RNAs, we identified two known miRNAs (miR166 and miR167), eight potential miRNAs, and ten putative heterochromatic siRNAs (hc-siRNAs). RNA gel blot analyses clearly showed that miR166 and miR167 were accumulated in the uninuclear pollen stage of anther development and remained until the tricellular pollen stage. Our cloning and RNA gel blot analyses of small RNAs led us to propose a possible function of small RNA-mediated gene regulation for the development of male gametes in rice.