it is well accepted that present - day mitochondria and chloroplasts resulted from the integration of living organisms into an ancestral mother cell 1 . during evolution , most genes originally encoded by the endosymbiont have been translocated to the nuclear genome . however , most processes of gene translocation are still unclear . in recent studies , many mitochondrial genes that have been translocated into the nuclear genome for instance , the respiratory mitochondrial gene cox2 , which is ubiquitously found in the mitochondrial genome , is absent in the mitochondrial genome of the cowpea but has been translocated to the nuclear genome 2 . in rice , an rps11-related sequence is present in the rice mitochondrial genome but it is non - functional because of an internal stop codon in the original reading frame . a functional rps11 gene has been identified in the rice nuclear genome and is well characterized 3 . more than 500 translocation events have been identified in which a mitochondrial sequence transferred into the rice nuclear genome 4 . the fate of the longest fragment , comprising 248 kb , identified in these events is further characterized in this study . the available rice genome database ( http://rgp.dna.affrc.go.jp/ ) and the blast programs in the riceblast blast search service ( http://riceblast.dna.affrc.go.jp/ ) were used in this study . the available rice genome database ( http://rgp.dna.affrc.go.jp/ ) and the blast programs in the riceblast blast search service ( http://riceblast.dna.affrc.go.jp/ ) were used in this study . dna sequences that have translocated from the mitochondrial genome to the nuclear genome have been identified at over 500 loci 4 . the longest of these sequences , a 248-kb mitochondrial fragment , was identified on chromosome 12 ( fig . the 248-kb mitochondrial dna fragment in the nuclear genome is designated ' 248-kb numtdna ' in this study . the sequence starts at position 20493130 and ends at 20741523 , corresponding to 71.2 - 71.7 centimorgans , on chromosome 12 . the 248-kb numtdna was contiguous across three overlapping bacterial artificial chromosome ( bac ) clones : al731738 , al713949 , and al732377 . in the 248-kb numtdna , the remaining 190-kb numtdna is of mitochondrial origin , corresponding to 38.79% of the mitochondrial genome . it has 99.68% identity with the original mitochondrial sequence 5 , strongly suggesting that the sequence transferred from the mitochondrial to the nuclear genome very recently . however , the arrangement of dna segments is very different in the 248-kb numtdna and the present mitochondrial genome . this suggests that the past mitochondrial dna conformation was quite different from the present conformation , rather than that dna fragments from different mitochondrial positions integrated into the same chromosomal locus or that dna rearrangements have occurred within the nuclear genome . if the mutation rate is 5 - 30  10 per site per year in the plant nucleus 6 , the translocation of the 190-kb numtdna is estimated to have happened about 1.0 - 6.4 x 10 years ago . our study and the previous arabidopsis study suggest that the plant nuclear genome is flexible enough to accept into chromosomes more than 190 kb of foreign dna sequence at a time . seven segments comprising 58 kb within the 248-kb numtdna are inferred to be of non - mitochondrial origin because no homology was identified between them and the present rice mitochondrial genomic sequence . four of these segments were found to be retrotransposon - related sequences in that they contain 3982-bp , 4228-bp , 3155-bp , and 3237-bp long terminal repeats ( ltr ) . the total lengths of the four retrotransposons are 10.7 kb , 11.3 kb , 12.1 kb , and 10.7 kb , respectively ( table 1 ) . one of them is a ty3-gypsy like retrotransposon on the basis of its structure 8 , but the other three are class i retrotransposons that had not yet been categorized . to determine the possible ancestral retrotransposons , we conducted ncbi blastx searches 9 using internal sequences from the three retrotransposons as queries . among the retrotransposons extracted from the analysis , we found several retrotransposons encoding full sets of proteins , including capsid - like protein , endonuclease , integrase , protease , reverse transcriptase , and rnase - h 8 . a comparison of the orders of these protein - encoding sequences strongly suggests that all the retrotransposons are ty3-gypsy like retrotransposon . therefore , we conclude that all four retrotransposons in the 248-kb numtdna were derived from a ty3-gypsy like retrotransposon . disruption of the three retrotransposons may have occurred during translocation , in the process of reverse transcription or integration . because the migration of the 190-kb numtdna into the nuclear genome was quite recent and no corresponding retrotransposon sequence was identified in the original mitochondrial sequence , it is highly likely that the transposition of the four retrotransposons into the numtdna occurred very recently . it also suggests that the four retrotransposons were active at the time of integration . in short , these retrotransposons integrated into the 190-kb numtdna since 1.0 - 6.4 x 10 years ago . tos17 is an endogenous copia - like retrotransposon of rice that is activated in tissue culture . the four retrotransposon segments are different from tos17 and are actually integrated into the numtdna , so they are possibly another kind of active retrotransposon , but their activation triggers are not known . an ncbi blast search 9 was conducted to determine the origins of the three remaining segments that were not mitochondrially derived ( table 1 ) . sequence unknown 1 ( 8719 bp ) contained 2003 bp and 606 bp of the rice mitochondrial and chloroplast sequences , respectively , whereas no information about the remaining 6110-bp sequence is available . sequences unknown 2 ( 1118 bp ) and unknown 3 ( 3436 bp ) are similar to maize mitochondrial sequences of 358 bp and 478 bp , respectively . these data suggest that the two sequences were present in the mitochondrial genome in the past but have been lost from the present form of the rice mitochondrial genome . in conclusion , the 248-kb numtdna is a good relic sequence with which to determine the past mitochondrial genome structure and the behavior of independent retrotransposons during evolution . dna segments of foreign origin , like numtdna , should make a considerable contribution to the study of genome evolution . dna sequences that have translocated from the mitochondrial genome to the nuclear genome have been identified at over 500 loci 4 . the longest of these sequences , a 248-kb mitochondrial fragment , was identified on chromosome 12 ( fig . the 248-kb mitochondrial dna fragment in the nuclear genome is designated ' 248-kb numtdna ' in this study . the sequence starts at position 20493130 and ends at 20741523 , corresponding to 71.2 - 71.7 centimorgans , on chromosome 12 . the 248-kb numtdna was contiguous across three overlapping bacterial artificial chromosome ( bac ) clones : al731738 , al713949 , and al732377 . in the 248-kb numtdna , the remaining 190-kb numtdna is of mitochondrial origin , corresponding to 38.79% of the mitochondrial genome . it has 99.68% identity with the original mitochondrial sequence 5 , strongly suggesting that the sequence transferred from the mitochondrial to the nuclear genome very recently . however , the arrangement of dna segments is very different in the 248-kb numtdna and the present mitochondrial genome . this suggests that the past mitochondrial dna conformation was quite different from the present conformation , rather than that dna fragments from different mitochondrial positions integrated into the same chromosomal locus or that dna rearrangements have occurred within the nuclear genome . if the mutation rate is 5 - 30  10 per site per year in the plant nucleus 6 , the translocation of the 190-kb numtdna is estimated to have happened about 1.0 - 6.4 x 10 years ago . our study and the previous arabidopsis study suggest that the plant nuclear genome is flexible enough to accept into chromosomes more than 190 kb of foreign dna sequence at a time . seven segments comprising 58 kb within the 248-kb numtdna are inferred to be of non - mitochondrial origin because no homology was identified between them and the present rice mitochondrial genomic sequence . four of these segments were found to be retrotransposon - related sequences in that they contain 3982-bp , 4228-bp , 3155-bp , and 3237-bp long terminal repeats ( ltr ) . the total lengths of the four retrotransposons are 10.7 kb , 11.3 kb , 12.1 kb , and 10.7 kb , respectively ( table 1 ) . one of them is a ty3-gypsy like retrotransposon on the basis of its structure 8 , but the other three are class i retrotransposons that had not yet been categorized . to determine the possible ancestral retrotransposons , we conducted ncbi blastx searches 9 using internal sequences from the three retrotransposons as queries . among the retrotransposons extracted from the analysis , we found several retrotransposons encoding full sets of proteins , including capsid - like protein , endonuclease , integrase , protease , reverse transcriptase , and rnase - h 8 . a comparison of the orders of these protein - encoding sequences strongly suggests that all the retrotransposons are ty3-gypsy like retrotransposon . therefore , we conclude that all four retrotransposons in the 248-kb numtdna were derived from a ty3-gypsy like retrotransposon . disruption of the three retrotransposons may have occurred during translocation , in the process of reverse transcription or integration . because the migration of the 190-kb numtdna into the nuclear genome was quite recent and no corresponding retrotransposon sequence was identified in the original mitochondrial sequence , it is highly likely that the transposition of the four retrotransposons into the numtdna occurred very recently . it also suggests that the four retrotransposons were active at the time of integration . in short , these retrotransposons integrated into the 190-kb numtdna since 1.0 - 6.4 x 10 years ago . tos17 is an endogenous copia - like retrotransposon of rice that is activated in tissue culture . the four retrotransposon segments are different from tos17 and are actually integrated into the numtdna , so they are possibly another kind of active retrotransposon , but their activation triggers are not known . an ncbi blast search 9 was conducted to determine the origins of the three remaining segments that were not mitochondrially derived ( table 1 ) . sequence unknown 1 ( 8719 bp ) contained 2003 bp and 606 bp of the rice mitochondrial and chloroplast sequences , respectively , whereas no information about the remaining 6110-bp sequence is available . sequences unknown 2 ( 1118 bp ) and unknown 3 ( 3436 bp ) are similar to maize mitochondrial sequences of 358 bp and 478 bp , respectively . these data suggest that the two sequences were present in the mitochondrial genome in the past but have been lost from the present form of the rice mitochondrial genome . in conclusion , the 248-kb numtdna is a good relic sequence with which to determine the past mitochondrial genome structure and the behavior of independent retrotransposons during evolution . dna segments of foreign origin , like numtdna , should make a considerable contribution to the study of genome evolution . schematic representation of the 248-kb mitochondrial sequence on chromosome 12 and the corresponding sequences on a master circle of rice mitochondrial genome . ( a ) three contiguous bac clones carrying mitochondrial sequences are indicated by bold lines . a black box and a circle represent a mitochondrial sequence and a centromere on chromosome 12 , respectively . ( b ) a presumptive mitochondrial master circle of the rice mitochondrial genome . sequences with strong similarity are shown in the same colors a model for the evolution of the 248-kb numtdna on rice chromosome 12 . step 2 - 2 , recombination events within the mitochondrial genome evolutionary information on the dna segments in the 248-kb numtdna positions on chromosome 12 ( chr12 ) and the mitochondrial ( mt ) genome and their sequence similarities are shown . 2 ) comparing one part of the ltr against the other part of the ltr in the same retrotransposon . * 3 ) ty3-gypsy like retrotransposon ; * 1 , * 2 , * 4 thought to be ty3-gypsy like retrotransposons
a 190-kb mitochondrial dna sequence interrupted by seven foreign dna segments was identified in rice chromosome 12 . 
 this fragment is the largest mitochondrial fragment translocated into the rice nuclear genome . 
 the sequence is composed of a 190-kb segment of mitochondrial origin corresponding to 38.79% of the mitochondrial genome , 45 kb comprising four segments of retrotransposon origin , and 13 kb comprising three segments of unknown origin . 
 the 190-kb sequence shows more than 99.68% similarity to the current mitochondrial sequence , suggesting that its integration into the nucleus was quite recent . 
 several sequences in the 190-kb segment have been rearranged relative to the current mitochondrial sequence , suggesting that the past and present arrangements of the mitochondrial genome differ . 
 the four retrotransposons show no mutual sequence similarity and are integrated into different locations , suggesting that their integration events were independent , frequent , and quite recent . 
 a fragment of the mitochondrial genome present in the nuclear genome , such as the 248-kb sequence characterized in this study , is a good relic with which to investigate the past mitochondrial genome structure and the behavior of independent retrotransposons during evolution .