Functional analysis of plastid DNA replication origins in tobacco by targeted inactivation

Mühlbauer, Stefan Klaus; Lössl, Andreas Günter; Tzekova, Lilia; Zou, Zhurong; Koop, Hans‐Ulrich

doi:10.1046/j.1365-313x.2002.01408.x

Cited by 44 publications

(37 citation statements)

References 31 publications

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“…In vitro replication analysis has revealed that the minimal sequences required for activity of single-ori ctDNA templates in tobacco is 82 bp for oriA and 243 bp for oriB (Kunnimalayaan and Nielsen, 1997b). There are conflicting reports on the effect of deletions of chloroplast oris (Muhlbauer et al, 2002;Kunnimalayaan and Nielsen, 1997b;Lu et al, 1996;Lugo et al, 2004). It is quite possible that disruption of an ori in vivo is not lethal if another mechanism can take over.…”

Section: Discussionmentioning

confidence: 99%

Stable transformation of the cotton plastid genome and maternal inheritance of transgenes

2004

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Chloroplast genetic engineering overcomes concerns of gene containment, low levels of transgene expression, gene silencing, positional and pleiotropic effects or presence of vector sequences in transformed genomes. Several therapeutic proteins and agronomic traits have been highly expressed via the tobacco chloroplast genome but extending this concept to important crops has been a major challenge; lack of 100 homologous species-specific chloroplast transformation vectors containing suitable selectable markers, ability to regulate transgene expression in developing plastids and inadequate tissue culture systems via somatic embryogenesis are major challenges. We employed a 'Double Gene/Single Selection (DGSS)' plastid transformation vector that harbors two selectable marker genes (aph A-6 and npt II) to detoxify the same antibiotic by two enzymes, irrespective of the type of tissues or plastids; by combining this with an efficient regeneration system via somatic embryogenesis, cotton plastid transformation was achieved for the first time. The DGSS transformation vector is at least 8-fold (1 event/2.4 bombarded plates) more efficient than 'Single Gene/Single Selection (SGSS)' vector (aph A-6; 1 event per 20 bombarded plates). Chloroplast transgenic lines were fertile, flowered and set seeds similar to untransformed plants. Transgenes stably integrated into the cotton chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. Cotton is one of the most important genetically modified crops (120 billion US dollars US annual economy). Successful transformation of the chloroplast genome should address concerns about transgene escape, insects developing resistance, inadequate insect control and promote public acceptance of genetically modified cotton.

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Section: Discussionmentioning

confidence: 99%

Stable transformation of the cotton plastid genome and maternal inheritance of transgenes

2004

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“…oriB1 is in the coding region of the essential gene ycf1, whose function is still not known (Drescher et al 2000). oriB2 is in the reading frame orf 350 and-unlike oriB1-it can be deleted from all plastome copies (Mü hlbauer et al 2002). It is not clear, if these differences between the oriBs reflect biological differences, e.g.…”

Section: Identity Of Fragment Endsmentioning

confidence: 99%

“…In addition, in suspension cultures of N. tabacum a different replication origin-also in the inverted repeats-was found (Takeda et al 1992). It was possible to delete both oriA sequences from all plastome copies and-in different lines-also one of the oriB sequences was completely removed (Mü hlbauer et al 2002). ptDNA replication in N. tabacum might therefore be possible from only one of the oriB elements.…”

Section: Introductionmentioning

confidence: 99%

Linear molecules of tobacco ptDNA end at known replication origins and additional loci

Scharff

Koop

2006

Plant Mol Biol

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Higher plant plastid DNA (ptDNA) is generally described as a double-stranded circular molecule of the size of the monomer of the plastid genome. Also, the substrates and products of ptDNA replication are generally assumed to be circular molecules. Linear or partly linear ptDNA molecules were detected in our present study using pulsed-field gel electrophoresis and Southern blotting of ptDNA restricted with 'single cutter' restriction enzymes. These linear DNA molecules show discrete end points which were mapped using appropriate probes. One possible explanation of discrete ends would be that they represent origins of replication. Indeed, some of the mapped ends correlate well with the known origins of replication of tobacco plastids, i.e. both of the oriA sequences and--less pronouncedly--with the oriB elements. Other ends correspond to replication origins that were described for Oenothera hookeri, Zea mays, Glycine max and Chlamydomonas reinhardtii, respectively, while some of the mapped ends were not described previously and might therefore represent additional origins of replication.

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“…OriA/B-independent replication of the spa19 PS1 genome is conceptually consistent with other examples. For example, deletion of oriA in tobacco chloroplasts had no obvious effect, but both copies of oriB could not be deleted (Muhlbauer et al 2002) and indeed, tobacco cpDNA appears to have multiple replication origins (Scharff and Koop 2007). We also note that OriA could not by itself promote maintenance of an extrachromosomal element in Chlamydomonas chloroplasts (Suzuki et al 1997).…”

Section: Discussionmentioning

confidence: 73%

Differential Replication of Two Chloroplast Genome Forms in Heteroplasmic Chlamydomonas reinhardtii Gametes Contributes to Alternative Inheritance Patterns

Nishimura

Stern

2010

Genetics

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Two mechanisms for chloroplast DNA replication have been revealed through the study of an unusual heteroplasmic strain of the green alga Chlamydomonas reinhardtii. Heteroplasmy is a state in which more than one genome type occurs in a mitochondrion or chloroplast. The Chlamydomonas strain spa19 bears two distinct chloroplast genomes, termed PS1 and PSÀ. PS1 genomes predominate and are stably maintained in vegetative cells, despite their lack of known replication origins. In sexual crosses with spa19 as the mating type plus parent, however, PS1 genomes are transmitted in only $25% of tetrads, whereas the PSÀ genomes are faithfully inherited in all progeny. In this research, we have explored the mechanism underlying this biased uniparental inheritance. We show that the relative reduction and dilution of PS1 vs. PSÀ genomes takes place during gametogenesis. Bromodeoxyuridine labeling, followed by immunoprecipitation and PCR, was used to compare replication activities of PS1 and PSÀ genomes. We found that the replication of PS1 genomes is specifically suppressed during gametogenesis and germination of zygospores, a phenomenon that also was observed when spa19 cells were treated with rifampicin, an inhibitor of the chloroplast RNA polymerase. Furthermore, when bromodeoxyuridine incorporation was compared at 11 sites within the chloroplast genome between vegetative cells, gametes, and rifampicin-treated cells by quantitative PCR, we found that incorporation was often reduced at the same sites in gametes that were also sensitive to rifampicin treatment. We conclude that a transcription-mediated form of DNA replication priming, which may be downregulated during gametogenesis, is indispensable for robust maintenance of PS1 genomes. These results highlight the potential for chloroplast genome copy number regulation through alternative replication strategies.

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Functional analysis of plastid DNA replication origins in tobacco by targeted inactivation

Cited by 44 publications

References 31 publications

Stable transformation of the cotton plastid genome and maternal inheritance of transgenes

Stable transformation of the cotton plastid genome and maternal inheritance of transgenes

Linear molecules of tobacco ptDNA end at known replication origins and additional loci

Differential Replication of Two Chloroplast Genome Forms in Heteroplasmic Chlamydomonas reinhardtii Gametes Contributes to Alternative Inheritance Patterns

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