The International Human Genome Sequencing Consortium completed the decoding of the human genome sequence in 2003. Readers will be aware of the paradigm shift which has occurred since then in the field of life science research. At last, mankind has been able to focus on a complete picture of the full extent of the genome, on which is recorded the basic information that controls all life. Meanwhile, another genome project, centered on Japan and known as the mouse genome encyclopedia project, was progressing with participation from around the world. Led by our research group at RIKEN, it was a full-length cDNA project which aimed to decode the whole RNA (transcriptome) using the mouse as a model. The basic information that controls all life is recorded on the genome, but in order to obtain a complete picture of this extensive information, the decoding of the genome alone is far from sufficient. These two genome projects established that the number of letters in the genome, which is the blueprint of life, is finite, that the number of RNA molecules derived from it is also finite, and that the number of protein molecules derived from the RNA is probably finite too. A massive number of combinations is still involved, but we are now able to understand one section of the network formed by these data. Once an object of study has been understood to be finite, establishing an image of the whole is certain to lead us to an understanding of the whole. Omics is an approach that views the information controlling life as finite and seeks to assemble and analyze it as a whole. Here, I would like to present our transcriptome research while making reference to our unique research strategy.
We used gametocidal (Gc) chromosomes 2C and 3CSAT to dissect barley 2H added to common wheat. The Gc chromosome induces chromosomal breakage resulting in chromosomal aberrations in the progeny of the 2H addition line of common wheat carrying the monosomic Gc chromosome. We conducted in situ hybridization to select plants carrying structurally rearranged aberrant 2H chromosomes and characterized them by sequential C-banding and in situ hybridization. We established 66 dissection lines of common wheat carrying single aberrant 2H chromosomes. The aberrant 2H chromosomes were of either deletion or translocation or complicated structural change. Their breakpoints were distributed in the short arm (2HS), centromere (2HC) and the long arm (2HL) at a rough 2HS/2HC/2HL ratio of 2:1:2. We conducted PCR analysis of the 66 dissection lines using 115 EST markers specific to chromosome 2H. Based on the PCR result, we constructed a physical or cytological map of chromosome 2H that were divided into 34 regions separated by the breakpoints of the aberrant 2H chromosomes. Forty-seven markers were present in 2HS and 68 in 2HL. We compared the 2H cytological map with a previously reported 2H genetic map using 44 markers that were used in common to construct both maps. The order of markers in the distal region was the same on both maps but that in the proximal region was somewhat contradictory between the two maps. We found that the markers distributed rather evenly in the genetic map were actually concentrated in the distal regions of both arms as revealed by the cytological map. We also recognized an EST-marker or gene-rich region in the 2HL interstitial region slightly to the telomere.
The plant chondriome confers a complex nature. The atp4 gene (formerly called orf25) of Aegilops crassa (CR) harbors the promoter sequence of the rps7 gene from common wheat (Triticum aestivum cv. Chinese Spring, CS). The rps7 gene of CR has the promoter sequence of CS atp6. The atp6 gene of CR contains an unknown sequence inside of its coding region. Since repeat sequences have been found around the breaking points, these structural alterations are most likely generated through homologous recombination. In this study, PCR analysis was performed to detect structural alterations in each of three lines: euplasmic lines of Ae. crassa, Chinese Spring, and alloplasmic Chinese Spring wheat with the cytoplasm of Ae. crassa ((cr)-CS). We found that each of these lines contained both genotypes, although mitochondrial genotypes of CR in Chinese Spring wheat and CS genotypes in Ae. crassa were still retained as minor fractions (less than 10%). On the other hand, CS mitochondrial gene frequencies in ((cr)-CS) were shown to be ca. 30%. SNP analysis after DNA sequencing of these genes indicated that minor types of all three mitochondrial genes in alloplasmic wheat contained the mitochondrial gene types from pollens. Since the frequencies of paternal mitochondrial gene types in F1 were about 20%, successive backcrossing increased the frequencies of paternal mitochondrial gene types to around 30% in alloplasmic wheat. Expression profiles of these mitochondrial genes were quantitatively analyzed by RT-PCR. Transcripts of paternal mitochondrial gene types were scarcely found. This suggests that minor fractions including paternal mitochondrial gene types are maintained and silenced in the descendants.
Among the genus Populus, the sections Populus (white poplar), Aigeiros Duby (black poplar) and Tacamahaca Spach contain many tree species of economical and ecological important properties. Two parental maps for the inter-specific hybrid population of Populus adenopoda × P. alba (two species of Populus section) were constructed based on SSR and SRAP markers by means of a two-way pseudo-test cross mapping strategy. The same set of SSR markers developed from the P. trichocarpa (belonging to Tacamahaca section) genome which were used to construct the maps of P. deltoides and P. euramericana (two species of Aigeiros section) was chosen to analyze the genotype of the experimental population of P. adenopoda × P. alba. Using the mapped SSR markers as allelic bridges, the alignment of the white and black poplar maps to each other and to the P. trichocarpa physical map was conducted. The alignment showed high degree of marker synteny and colinearity and the closer relationship between Aigeiros and Tacamahaca sections than that of Populus and Tacamahaca. Moreover, there was evidence for the chromosomal duplication and inter-chromosomal reorganization involving some poplar linkage groups, suggesting a complicated course of fission or fusion in one of the lineages. A poplar consensus map based on the comparisons could be constructed will be useful in practical applications including marker assisted selection.
Mterfd2 is a component of mitochondria transcription termination factor (MTERF) family which belongs to the MTERF4 subfamily. In this report, we characterized the expression profile of mouse Mterfd2 during embryogenesis by in situ hybridization (ISH), quantitative real-time PCR (qRT-PCR) and northern blot. The whole mount ISH at E9.5, E10.5 and E11.5 showed that Mterfd2 was dynamically expressed in the brain. Besides, at E9.5 and E10.5 stages, Mterfd2 was persistently expressed in the lateral plate mesoderm and heart; at E10.5 and E11.5 stages, it showed an abundant expression in the limb buds. The tissue ISH of E13.5 and E15.5 suggested that Mterfd2 was ubiquitously expressed, and has the higher expression in the forebrain, diencephalon, midbrain, spinal cord, dorsal root ganglion, tongue, lung, liver and kidney. This ubiquitous expression profile in the late embryogenesis was further confirmed by qRT-PCR and northern blot at E12.5, E15.5 and E18.5 stages. Besides, the results of co-location of EGFP-Mterfd2 fusion protein indicated that Mterfd2 was targeted to the mitochondria. Collectively, these data suggested that Mterfd2 showed a dynamic expression pattern during embryogenesis. It might play an important role in the organ differentiation which was probably resulted from its role in the mitochondrial transcription regulation.
DYX1C1 is a candidate gene for developmental dyslexia and has three alternative pre-mRNA spliced forms in the human genome. One of the transcripts contains an HERV-H LTR that could affect the expression level of DYX1C1. We speculate that the HERV-H LTR integrated into the DYX1C1 locus in the catarrhine lineage after its divergence from the platyrrhine lineage. Reverse transcription-PCR of the HERV-H LTR-related transcript produced four alternative forms from several human tissues. All of alternative forms were also identified in various rhesus macaque tissues. Through sequencing analysis of various primate DNA samples, we found that a part of the HERV-H LTR sequence was duplicated within the DYX1C1 exon 9 only in catarrhines. However, the duplication event did not cause frameshift mutation of the DYX1C1 transcript. Taken together, this HERV-H LTR insertion into DYX1C1 has contributed to transcript diversification of DYX1C1 during primate evolution.