The most successfully studied in the Chlamydomonas alga. C. reinhardtii has only one chloroplast with about 80 cpDNA molecules of 196 kbp each. The transmission of the cpDNA genome is largely uniparental, i.e., inherited most commonly through the mt⁺ (comparable to egg) cytoplasm although in 1–10% of the cases biparental transmission may take place (alfalfa, Oenothera). The chloroplast nucleoid (DNA) transmitted by the mt⁻ mate is usually completely digested in Chlamydomonas within 10 minutes after zygote formation, whereas the mitochondrial nucleoid still remained intact. Specially, e.g., in conifers, uniparental male transmission may also exist. The uniparental mt⁺ transfer may sometimes be spurious in cases when the coding of the subunits of a particular protein is under the control of nuclear and organelle genes, respectively. In diploid vegetative zygotes of Chlamydomonas the biparental transmission of the extranuclear genes is most likely. Incubation in dark or postponing the meiosis by nitrogen starvation, however, favors uniparental transmission (see Fig. C78).

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In higher plants, the transmission of the plastid is usually through the female but biparental or only male transmission also occurs. The plastid nucleoids may be eliminated from, or degraded in, the sperms during the first or second nuclear division in the pollen, or they are left behind when the generative nucleus enters the egg.

In tobacco the frequency of transmission of transgenes though the pollen into the cotyledons of F1 seedlings was 1.58 × 10^--5 (at 100% cross-fertilization); transmission into the shoot apical meristem was significantly lower, 2.86 × 10^--6 (Ruf S et al 2007 Proc Natl Acad Sci USA 104:6998). In another experiment, plastids from the transgenic plastids of the male tobacco were transmitted (10^--4–10^--5) frequencies indicating again that the transgene can be reasonable well contained if it is carried by the plastids rather than by the nucleus (see Fig. C79). It was an interesting observation that the entire plastome (not only a fragment of it) and also the mitochondria were simultaneously transmitted at this rate (Svab Z, Maliga P 2007 Proc Natl Acad Sci USA 104:7003).

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The most common algal mutations (at different loci) require acetate as the carbon source because they cannot fix CO₂. Antibiotic resistance (or antibiotic dependent) mutations involve the rRNA or ribosomal proteins. Fluorodeoxyuridine is a specific mutagen for cpDNA. Arsenate and metronidazole are selective for nonphotosynthetic mutations. Photosynthesis defective mutations (nuclear or cpDNA) can also be screened under long-wave-length UV. Also, in higher plants, streptomycin, spectinomycin (16S rRNA) lincomycin (23S rRNA), etc., resistance mutations could be induced and isolated.

The partial inactivation of the mt⁺ cells by UV permits the transmission of cpDNA genes by the mt^- cells and this makes possible recombinational studies. However, some progeny cells are heteroplasmic even after 3 divisions of the zygote. In the cpDNA, only closely situated genes display linkage with about 1 kb/map unit. Mapping is practical by physical methods: either by deletional analysis or by positional cloning, or in interspecific crosses when RFLP exists by co-segregation with restriction endonuclease fragments (C. eugametos x C. moewusii). The nature of the recombination map was hotly debated until it turned out that genes within the inverted repeats map as if the area would be linear, whereas a recombination of genes outside this region reflects the circular nature of the cpDNA. A single recombination between two circular molecules may result in a cointegrate. Recombination of cpDNA genes in higher plants also occurs albeit apparently quite rarely. The chloroplasts usually do not “synapse.” Within cells of interspecific (intergeneric) somatic fusion in the Solanaceae recombination of antibiotic resistance markers has been demonstrated.

Co-segregation of mitochondrial traits (cytoplasmic male sterility and chloroplast antibiotic markers) was also shown. Transforming of appropriate genes (Atrazine resistance) into the cell nucleus, using T-DNA vectors, may alter chloroplast functions. Transformation—using biolistic procedures—of cpDNA can be accomplished at high frequencies (up to 1 × 10^-4) in Chlamydomonas and higher plants. The insertion takes place around the passenger and vector junctions and the insert replaces the resident copy in the nucleoid.

The insertion takes place by site-specific recombination and thus it is nonrandom. In tobacco the integration may be the outcome of multiple recombinational events. Co-transformation of antibiotic resistance genes situated in the inverted repeats and photosynthetic genes (situated in the unique regions) can be accomplished by the simultaneous use of two vectors. Alternatively, the bacterial aadA gene (that detoxifies antibiotics) is used. The gene is equipped by cpDNA transcription and translation elements and surrounded by the appropriate target sequences. Whether or not the transformation involves correction of a resident gene or insertion of a foreign gene (e.g., uidA [glucuronidase]), the integration requires homologous recombination within the flanking sequences. Transformation has also been achieved by direct transfer and integration of the cloned gene into protoplasts in the presence of polyethylene glycol or by the biolistic methods. Under selective conditions sorting out of the transgene takes place rapidly.

The segregation and sorting out of chloroplast genes were subjected to analysis by the methods of population genetics and computer simulation. The biological observations do not seem to support the stochastic models of sorting out.

Mobile genetic elements as Group I introns, encoding the I-CreI (or I-CeuI) endonuclease and locating in the large ribosomal subunit gene or in the cytochrome b gene (I-CsmI endonuclease) in mt ⁺ (mating type) plastid nucleoids, have been found in different Chlamydomonas chloroplasts. The I-CreI endonuclease has a recognition site of 24 bp. These mobile elements resemble those of I-Sce in the mitochondria of yeast.

The promoter regions of the cpDNA genome are similar to that of prokaryotes. There are generally 10 nucleotides between the TATA box (TATAAT or longer) and the first translated codon and again 17–19 bases separate the TATA box from the 5′-TTGACA promoter consensus at about -35. Internal and further upstream promoters have also been identified. Many genes are transcribed into polycistronic RNAs. In spinach, 18 major RNAs were made from a single polycistronic transcript. The 3′ termini of the transcripts generally contain inverted repeats that have probably only some processing and/or stabilizing functions along with some 5′-untranslated sequences. Higher plants appear to have a second DNA-dependent RNA polymerase, which is encoded by the nucleus (Liere K et al 2004 Nucleic Acids Res 32:1159). This polymerase transcribes a different set of chloroplast genes. Binding proteins (3′) seem to be involved in the processing of the RNA. Despite common ancestry with bacterial translation, chloroplast translation is more complex and involves positive regulatory mRNA elements and a host of requisite protein translation factors that do not have counterparts in bacteria. Previous proteomic analyses of the chloroplast ribosome identified a significant number of chloroplast-unique ribosomal proteins that expand upon a basic bacterial 70S-like composition (Manuell L et al 2007 PloS Biol 5[8]:e209). Some observations indicate that polycistronic transcripts may bind to the ribosomes and translated without processing, perhaps with reduced efficiency. Translation of the chloroplast mRNA appears to be light-regulated. Apparently, an activator protein binds to the upstream untranslated region of the mRNA and the regulation is mediated through the redox state of this protein. Endonucleolytic processing of the transcripts may provide alternative leader sequences and binding sites for transcription factors. Ribosomal proteins may exert activation also by induction and modulation of translation. The chloroplast mRNAs are not capped and the initiation of transcription is regulated in a manner similar to that in prokaryotes. In the untranslated upstream regions (UTR) there are binding sites for nuclearly encoded proteins that regulate transcription. Other proteins may bind to the 3′ downstream sequences. Nuclear proteins mediate translational control (Choquet Y, Wollman F-A 2002 FEBS Lett 529:39). The chloroplast DNA most commonly encodes ∼30 tRNAs. In the plastids of nongreen plants, the tRNA gene number is reduced to about half.  chloroplasts,  chloroplast mapping,  physical mapping,  ctDNA,  RFLP,  cointegration,  deletion mapping,  biolistic transformation,  transformation of organelles,  β−glucuronidase,  mutation in cellular organelles,  heteroplastidic,  sorting out,  introns,  twintrons,  maturase,  mitochondrial genetics,  transcription,  ribosomes,  σ,  translation,  mating type,  polycistronic,  promoter,  nucleoid,  binding protein,  processing,  redox reaction,  RNA editing,  endosymbiont theory,  nucleomorph,  metronidazole; Sugiura M et al 1998 Annu Rev Genet 32:437; Jarvis P 2001 Curr Biol 11:R307; Hagemann R 2000 J Hered 91:435; Rodermel S 2001 Trends Plant Sci 6:471; Ogihara Y et al 2002 Mol Genet Genomics 266:740; genome database: http://chloroplast.cbio.psu.edu/.