Chloroplast recombination

Genetic data have been corroborated by physical evidence for chloroplast recombination (, ). These data suggested that, given an opportunity for chloroplast mixing, recovery of chloroplast recombinants is feasible in flowering plants. 

Plant cell culture techniques have provided the tools to enable successful recovery of chloroplast recombinants. These include utilization of selectable chloroplast markers such as resistance to the emtibiotics streptomycin and lincomycln (10) and efficient methods for somatic cell fusion that enable the production of large numbers of cells with mixed chloroplast populations (33). 

Transcriptional inhibitors could be used simultaneously. Rifampicin blocks chloroplast and mitocondrian RNA synthesis [23, 24], while tagetitoxin is a very specific inhibitor of chloroplast RNA polymerase [25]. Treatment with these antibiotics does not inhibit Rubisco SSU synthesis since the promoter is part of the nuclear genome, while the cytosolic ribosomes are not affected by streptomycin. Therefore SSU promoters can be used to drive transgene expression and facilitate the accumulation of recombinant proteins. Expressed proteins are targeted to a suitable cellular compartment, such as the cytoplasm, apoplastic space or chloroplast, depending on the nature of the protein. 

The coding sequences of a- and P-globins of human hemoglobin have been fused to the sequence of the chloroplastic transit peptide of the small subunit of Rubisco. These proteins were then coexpressed in transgenic tobacco plants, resulting in the production of a functional form of tetra-meric hemoglobin. The results demonstrate that a complex multimeric protein such as recombinant human hemoglobin can be obtained from tobacco in a functional form. 

Li, H.Y., Ramalingam, S., and Chye, M.L. (2006). Accumulation of recombinant SARS-CoCV spike protein in plant cytosol and chloroplasts indicate potential for development of plant-derived oral vaccines. Exp. Biol. Med. (Maywood) 231(8) 1346-1352. 

While a DNA molecule may exist as a straight rod, the two ends are often covalently joined. Thus, the chromosomes of E. coli and of other bacteria are single closed circles. Circular DNA molecules are also found in mitochondria, chloroplasts, and many viruses. Further complexity arises from the fact that the circles of DNA are sometimes interlocked in chainlike fashion (catenated). An unusual example of this phenomenon is the presence of thousands of small catenated DNA circles in the single mitochondrion of a trypanosome (Fig. 5-16).183 Sometimes circular DNA is knotted as in Fig. 5-17.184-186 Knots and catenanes often appear as intermediate forms during replication and recombination, especially involving circular DNA.187 188... 

Four basic strategies for recombinant protein expression in plants are transient expression, stable nuclear transformed plants, chloroplast transformed plants, and suspension cultures derived from stable transgenic lines. 

At present it is debatable whether all chloroplastic MIPS are splice variant of the cytosolic form (Lackey et al., 2003) or they are descendants of the cyanobacterial form. It might even be possible to find more than one chloroplastic MIPS enzyme with different origins. However, until the chloroplastic gene is cloned and the recombinant protein is characterized for its enzymatic properties, the debate remains open with lively and exciting discoveries ahead. 

Yoshimura, K., Ishikawa, T., Nakumura, Y., Tamoi, M., Takeda, T., Tada, T., Nishimura, K., and Shigeoka, S., 1998, Comparative study on recombinant chloroplastic and cytosolic ascorbate peroxidase isozymes of spinach. Arch. Biochem. Biophys. 353 55n63. 

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