Variable copy number of macronuclear DNA molecules in Tetrahymena

In Tetrahymena, the DNA of the macronucleus exists as very large (100 to 4,000-kb) linear molecules that are randomly partitioned to the daughter cells during cell division. This genetic system leads directly to an assortment of alleles such that all loci become homozygous during vegetative growth. Apparently, there is a copy number control mechanism operative that adjusts the number of each macronuclear DNA molecule so that macronuclear DNA molecules (with their loci) are not lost and aneuploid death is a rare event. In comparing Southern analyses of the DNA from various species of Tetrahymena using histone H4 genes as a probe, we find different band intensities in many species. These differences in band intensities primarily reflect differences in the copy number of macronuclear DNA molecules. The variation in copy number of macronuclear DNA molecules in some species is greater than an order of magnitude. These observations are consistent with a developmental control mechanism that operates by increasing the macronuclear copy number of specific DNA molecules (and the genes located on these molecules) to provide the relatively high gene copy number required for highly expressed proteins. © 1992 Wiley-Liss, Inc.     

Identification of the entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice

Summary The entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice (Oryza sativa cv. Nipponbare) was identified using clone banks that cover the chloroplast and mitochondrial genomes. The mitochondrial fragments that were homologous to chloroplast DNA were mapped and sequenced. The nucleotide sequences around the termini of integrated chloroplast sequences in the rice mtDNA revealed no common sequences or structures that might enhance the transfer of DNA. Sixteen chloroplast sequences, ranging from 32 bases to 6.8 kb in length, were found to be dispersed throughout the rice mitochondrial genome. The total length of these sequences is equal to approximately 6% (22 kb) of the rice mitochondrial genome and to 19% of the chloroplast genome. The transfer of segments of chloroplast DNA seems to have occurred at different times, both before and after the divergence of rice and maize. The mitochondrial genome appears to have been rearranged after the transfer of chloroplast sequences as a result of recombination at these sequences. The rice mitochondrial DNA contains nine intact tRNA genes and three tRNA pseudogenes derived from the chloroplast genome.     

Variations in chloroplast DNA from rice (Oryza sativa): differences between deletions mediated by short direct-repeat sequences within a single species

In a previous study, we compared chloroplast DNAs (ctDNAs) from four species ofOryza and detected two independent deletions of DNA fragments in the ctDNAs (Kanno and Hirai 1992a). These deletions were genotype-specific variations. Since short direct-repeat sequences were detected at the borders of both deletions, the deletions were apparently the result of intramolecular recombination mediated by these direct-repeat sequences. In the present study, we examined whether or not this type of variation exists within a single species. Ishii et al. demonstrated three types of ctDNA inO. Sativa (1988), and the source of the variations that they identified seemed to be deletions. We determined the precise locations of the deletions and the sequences around them. As expected, our results showed that these variations were the results of deletions that were mediated by short direct-repeat sequences. While the deletions that had been found previously were located on spacer regions, those found in this study were located within open reading frames (ORFs). Northern hybridization analysis showed that one of the ORFs was-transcribed. In the case of this deletion, the amino acid sequence encoded by the C-terminal region of the ORF was altered and the short inverted-repeat sequences downstream of the ORF were deleted. In addition, there were other short inverted-repeat sequences downstream of the altered ORF.     

Homologies to chloroplast DNA in the nuclear DNA of a number of Chenopod species

Summary Sequences homologous to chloroplast (ct)DNA have been found in nuclear DNA in five species of the Chenopodiaceae, extending the earlier observations of promiscuous DNA in Spinacia oleracea (Timmis and Scott 1983). Using the 7.7 kbp spinach ctDNA Pst I fragment as a hybridization probe, several separately located homologies to ctDNA were resolved in the nuclear DNA of Beta vulgaris, Chenopodium quinoa, and Enchylaena tomentosa. In Chenopodium album and Atriplex cinerea the major region of homology was to a nuclear Eco RI fragment (6 kbp) indistinguishable from that in ctDNA. These homologies may therefore involve larger tracts of ctDNA because the same restriction sites are apparently retained in the nucleus. This suggests that in these latter two species there is a contrasting, more homogeneous arrangement of ctDNA transpositions in the nucleus.     

Detection of somaclonal variation in cultured rice cells using digoxigenin-based random amplified polymorphic DNA

A procedure for the non-radioactive detection of random amplified polymorphic DNA (RAPD) was developed and designated as digoxigenin (DIG)-based RAPD. Using this procedure, we analyzed somaclonal variation in cultured cells of rice. Somaclonal variation was found to increase with culture age. More than 50 polymorphic fragments were identified with the four primers tested. Random sequencing of 10 clones generated one intron, one 5′-noncoding, and eight non-redundant expressed sequences. A database search for homology showed that the eight exon sequences displayed a significant similarity to sequences already stored in EMBL, GenBank and DDBJ. The sources of the known genes ranged from microorganism to human, including three rice genes. The results showed that somaclonal variation might have occurred in transfer RNA, ribosomal protein, and other genes during cell culture. Received: 14 November 1997 / Revision received: 21 August 1998 / Accepted: 31 August 1998     

Quantification of total genomic DNA and selected repetitive sequences reveals concurrent changes in different DNA families in indica and japonica rice

This paper describes a fluorescence in situ hybridization (FISH) analysis of three different repetitive sequence families, which were mapped to mitotic metaphase chromosomes and extended DNA fibers (EDFs) of the two subspecies of rice (Oryza sativa), indica and japonica (2n=2x=24). The repeat families studied were (1) the tandem repeat sequence A (TrsA), a functionally non-significant repeat; (2) the [TTTAGGG]_n telomere sequence, a non-transcribed, tandemly repeated but functionally significant repeat; and (3) the 5S ribosomal RNA (5S rDNA). FISH of the TrsA repeat to metaphase chromosomes of indica and japonica cultivars revealed clear signals at the distal ends of twelve and four chromosomes, respectively. As shown in a previous report, the 17S ribosomal RNA genes (17S rDNA) are located at the nucleolus organizers (NORs) on chromosomes 9 and 10 of the indica cultivar. However, the japonica rice lacked the rDNA signals on chromosome 10. The size of the 5S rDNA repeat block, which was mapped on the chromosome 11 of both cultivars, was 1.22 times larger in the indica than in the japonica genome. The telomeric repeat arrays at the distal ends of all chromosome arms were on average three times longer in the indica genome than in the japonica genome. Flow cytometric measurements revealed that the nuclear DNA content of indica rice is 9.7% higher than that of japonica rice. Our data suggest that different repetitive sequence families contribute significantly to the variation in genome size between indica and japonica rice, though to different extents. The increase or decrease in the copy number of several repetitive sequences examined here may indicate the existence of a directed change in genome size in rice. Possible reasons for this phenomenon of concurrent evolution of various repeat families are discussed. Received: 9 August 1999 / Accepted: 29 December 1999     

Variable copy number of macronuclear DNA molecules in Tetrahymena.

In Tetrahymena, the DNA of the macronucleus exists as very large (100 to 4,000-kb) linear molecules that are randomly partitioned to the daughter cells during cell division. This genetic system leads directly to an assortment of alleles such that all loci become homozygous during vegetative growth. Apparently, there is a copy number control mechanism operative that adjusts the number of each macronuclear DNA molecule so that macronuclear DNA molecules (with their loci) are not lost and aneuploid death is a rare event. In comparing Southern analyses of the DNA from various species of Tetrahymena using histone H4 genes as a probe, we find different band intensities in many species. These differences in band intensities primarily reflect differences in the copy number of macronuclear DNA molecules. The variation in copy number of macronuclear DNA molecules in some species is greater than an order of magnitude. These observations are consistent with a developmental control mechanism that operates by increasing the macronuclear copy number of specific DNA molecules (and the genes located on these molecules) to provide the relatively high gene copy number required for highly expressed proteins.     

Duplication and DNA segmental loss in the rice genome: implications for diploidization

* Large-scale duplication events have been recently uncovered in the rice genome, but different interpretations were proposed regarding the extent of the duplications. * Through analysing the 370 Mb genome sequences assembled into 12 chromosomes of Oryza sativa subspecies indica, we detected 10 duplicated blocks on all 12 chromosomes that contained 47% of the total predicted genes. Based on the phylogenetic analysis, we inferred that this was a result of a genome duplication that occurred c. 70 million years ago, supporting the polyploidy origin of the rice genome. In addition, a segmental duplication was also identified involving chromosomes 11 and 12, which occurred c. 5 million years ago. * Following the duplications, there have been large-scale chromosomal rearrangements and deletions. About 30-65% of duplicated genes were lost shortly after the duplications, leading to a rapid diploidization. * Together with other lines of evidence, we propose that polyploidization is still an ongoing process in grasses of polyploidy origins.     

Three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles

The cytological sequence of senescence-related changes in coleoptiles of rice (Oryza sativa L. cv. Nippon-bare) was studied using fluorescence and electron microscopy. The coleoptiles reach full size 3 d after sowing, then rapidly senesce and wither completely by day 7. The interveinal region in cross-sections taken 1 mm from the tip of the coleoptile was selected for this analysis. Fluorescence microscopy using samples embedded in Technovit 7100 resin, electron microscopy and immunoelectron microscopy using DNA-specific antibodies were used to elucidate the sequence of senescence-related events. These occur in the following order: (i) degradation of the chloroplast DNA (cpDNA); (ii) condensation of the nucleus in conjunction with a decrease in the size of the dense-chromatin region, shrinkage of the chloroplast, degradation of ribulose-1, 5-bisphosphate carboxylase/oxygenase, dilation of the thylakoid membranes, increase in size and number of osmiophilic globules, condensation of the cytoplasm; (iii) disorganization of the nucleus, degeneration of the tonoplast; (iv) complete loss of the cytoplasmic components, distortion of the cell wall, invasion of microorganisms into the intercellular spaces and ultimately into the cell itself. The mitochondria maintain their ultrastructural integrity and a constant level of mitochondrial DNA throughout senescence. In young mesophyll cells, invagination of the tonoplast into the vacuole frequently occurs. This occasionally includes cytoplasmic material, which is digested in the vacuole as senescence proceeds. Immunoelectron microscopy suggests that cpDNA degradation involves rough digestion first, rather than rapid, direct decomposition of the DNA into nucleotides. The fragmented cpDNA is then dispersed throughout the chloroplast and cytoplasm. Received: 9 April 1998 / Accepted: 11 June 1998     

Molecular analysis of organelle DNA of different subspecies of rice and the genomic stability of mtDNA in tissue cultured cells of rice

Summary Chloroplast (ct) and mitochondrial (mt) DNAs were isolated from two subspecies of rice (Oryza sativa), japonica (Calrose 76) and indica (PI353705) and compared by restriction endonuclease fragment pattern analysis. Similarly, PI353705 (A5) mtDNA was also compared with the mtDNA of its long term tissue cultured line, BL2. Variation in the ctDNA of the 2 subspecies was detected with two (AvaI and BglI) of the 11 restriction endonucleases tested, whereas their mtDNAs showed considerable variation when restricted by PstI, BamHI, HindIII and XhoI endonucleases. Thus, the chloroplast DNA was more highly conserved than the mtDNA in the subspecies comparisons. Only minor variation was observed between the restriction endonuclease patterns of the mtDNAs of BL2 and A5. Southern blots of mtDNA were hybridized with heterologous probes from maize and spinach organelle genes. Differences were found in the hybridization patterns of the two subspecies for six of the eight (mitochondrial and chloroplast) probes tested. Two of the seven (mitochondrial) probes (coxII and 26S rRNA) detected tissue culture generated variation in mtDNA. The relative values of restriction endonuclease and hybridization patterns for studying phylogenetic and genetic relationships in rice are discussed.Florida Agricultural Experiment Station Journal Series No. 8807. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable     

A contiguous sequence in spinach nuclear DNA is homologous to three separated sequences in chloroplast DNA

Summary A 3.4-kbp nuclear (n) DNA sequence has greater than 99% sequence homology to three segments of the chloroplast (cp) genes rps2, psbD/C, and psaA respectively. Each of these cpDNA segments is less than 3 kbp in length and appears to be integrated, at least in part, into several (>5) different sites flanked by unique sequences in the nuclear genome. Some of these sites contain longer homologies to the particular genes, while others are only homologous to smaller parts of the cp genes. Both the cpDNA fragments found in the nuclear genome and their flanking nDNA sequences are invested with short repeated A-T rich sequences but, apart from a hexanucleotide sequence and a palindromic sequence identified near each recombination point, there is no obvious structure that can suggest a mechanism of DNA transfer from the chloroplast to the nucleus in spinach.     

DNA uptake by imbibition and expression of a foreign gene in rice

Uptake of DNA by imbibition of dry and viable rice ( Oryza sativa L.) embryos from a DNA solution and expression of a foreign gene were detected using two different vectors contaíning gusA (β-glucuronidase) and hpt (hygromycin phosphotransferase) as reporter genes. The frequency of transient expression of gusA and hpt genes using the CaMV35S promoter was about 30 to 50%. The main sites of gusA gene expression were meristems of roots and vascular bundles of leaves. Also, DNA uptake, integration and expression of the hpt gene in selected rice were investigated by various PCR methods and Southern blot analysis of genomic DNA. It was shown that the hygromycin phosphotransferase (HPT) DNA was present in the rice genome in an integrated form and not as a plasmid form.     

Biochemical properties of a plastidial DNA polymerase of rice

Plastids are organelles unique to plant cells and are responsible for photosynthesis and other metabolic functions. Despite their important cellular roles, relatively little is known about the mechanism of plastidial DNA replication and repair. Recently, we identified a novel DNA polymerase in Oryza Sativa L. (OsPOLP1, formerly termed OsPolI-like) that is homologous to prokaryotic DNA polymerase Is (PolIs), and suggested that this polymerase might be involved in plastidial DNA replication and repair. Here, we propose to rename the plant PolI homologs as DNA polymerase π (POLP), and investigate the biochemical properties of full-length OsPOLP1. The purified OsPOLP1 elongated both DNA and RNA primer hybridized to a DNA template, and possessed a 3′ exonuclease activity. Moreover, OsPOLP1 displayed high processivity and fidelity, indicating that this polymerase has the biochemical characteristics appropriate for DNA replication. We found that POLPs have two extra sequences in the polymerase domain that are absent in prokaryotic PolIs. Deletion of either insert from OsPOLP1 caused a decrease in DNA synthetic activity, processivity, and DNA binding activity. In addition, OsPOLP1 efficiently catalyzed strand displacement on nicked DNA with a 5′-deoxyribose phosphate, suggesting that this enzyme might be involved in a repair pathway similar to long-patch base excision repair. These results provide insights into the possible role of POLPs in plastidial DNA replication and repair. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles

The coleoptile of rice (Oryza sativa L. cv. Nippon-bare) emerges from an imbibed seed on day 2 after sowing. Then, it matures and senesces rapidly. For analysis of the senescence pattern within individual coleoptiles, we monitored the distribution of chlorophyll (Chl) in entire coleoptiles and in cross-sections of coleoptiles by recording the autofluorescence of Chl. Degradation of Chl was apparent at the tip of the margins of opened-out coleoptiles on day 4, when the overall levels of soluble protein and Chl per coleoptile had reached maximum values. Then, senescence proceeded from the tip to the base and from the inner mesophyll cells towards the outer epidermis, excluding tissues along vascular bundles. Further analysis of cellular senescence using samples embedded in Technovit 7100 resin revealed that the senescence of each green mesophyll cell followed an identical program, which consisted of the following steps: (i) degradation of chloroplast DNA; (ii) condensation of the nucleus, decrease in the size of chloroplasts, degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase and chloroplast inner membranes; (iii) disorganization of the nucleus; (iv) complete loss of cellular components, distortion of the cell wall. Although the timing of each step and the rate at which each step was completed differed among cells of different locations within the coleoptile, this sequence was observed in all mesophyll cells in the coleoptile. Received: 31 July 1997 / Accepted: 28 October 1997     

Quantification of mitochondrial DNA copy number: Pre-analytical factors

Mitochondrial DNA (mtDNA) content is important for understanding many cellular processes. Several pre-analytical factors, from sample collection to DNA extraction can affect measurement of mtDNA copy number. In the present study, whole blood samples yielded a higher mtDNA copy number than buffy coat samples. mtDNA content is affected by the cell separation method used and the time between blood withdrawal and cell separation. Thus, reference values must be established with the same type of sample. As to the DNA isolation and purification method, the manual phenol method can give randomly false high values. The QIAamp DNA Mini Kit provided the most highly reproducible mtDNA/nDNA yield.     

Origins of weedy rice revealed by polymorphisms of chloroplast DNA sequences and nuclear microsatellites

Conspecific weeds that permanently infest worldwide agroecosystems are evolved from their crop species. These weeds cause substantial problems for crop production by competing for resources in agricultural fields. Weedy rice represents such a conspecific weed infesting rice ecosystems, and causing tremendous rice yield losses owing to its strong competitiveness and abundant genetic diversity, likely resulted from its complex origins. Here, we report the use of chloroplast DNA (cpDNA) fingerprints to determine whether weedy rice is evolved from its wild (exo‐feral) or cultivated (endo‐feral) rice progenitor as the maternal donor in recent hybridization events. In addition, we also applied nuclear simple sequence repeat (SSR) markers to confirm the exo‐feral or endo‐feral origins of weedy rice accessions determined by the cpDNA fingerprints. We found that the studied weedy rice accessions evolved either from their wild or cultivated rice progenitor, as the maternal donor, based on the cpDNA network and structure analyses. Combined analyses of cpDNA and nuclear SSR markers indicated that a much greater proportion of weedy rice accessions had the endo‐feral origin. In addition, results from the genetic structure of nuclear SSR markers indicated that weedy rice accessions from the endo‐feral pathway are distinctly associated with either indica or japonica rice cultivars, suggesting their complex origins through crop–weed introgression. The complex pathways of origin and evolution could greatly promote genetic diversity of weedy rice. Therefore, innovative methods should be developed for effective weedy rice control.     

Number matters: control of mammalian mitochondrial DNA copy number

Regulation of mitochondrial biogenesis is essential for proper cellular functioning. Mitochondrial DNA （mtDNA） depletion and the resulting mitochondrial malfunction have been implicated in cancer, neurodegeneration, diabetes, aging, and many other human diseases. Although it is known that the dynamics of the mammalian mitochondrial genome are not linked with that of the nuclear genome, very little is known about the mechanism of mtDNA propagation. Nevertheless, our understanding of the mode of mtDNA replication has ad- vanced in recent years, though not without some controversies. This review summarizes our current knowledge of mtDNA copy number control in mammalian cells, while focusing on both mtDNA replication and turnover. Although mtDNA copy number is seemingly in excess, we reason that mtDNA copy number control is an important aspect of mitochondrial genetics and biogenesis and is essential for normal cellular function.