Retrocyclin-101 (RC101) and Protegrin-1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections, especially those caused by the HIV-1 or sexually-transmitted bacteria. Because of their antimicrobial activity and complex secondary structures, they have not yet been produced in microbial systems and their chemical synthesis is prohibitively expensive. Therefore, we created chloroplast transformation vectors with the RC101 or PG1 coding sequence, fused with GFP to confer stability, furin or Factor Xa cleavage site to liberate the mature peptide from their fusion proteins and a His-tag to aid in their purification. Stable integration of RC-101 into the tobacco chloroplast genome and homoplasmy were confirmed by Southern blots. RC-101 and PG1 accumulated up to 32-38% and 17~26% of the total soluble protein. Both RC-101 and PG1 were cleaved from GFP by corresponding proteases in vitro and Factor Xa like protease activity was observed within chloroplasts. Confocal microscopy studies showed location of GFP fluorescence within chloroplasts. Organic extraction resulted in 10.6 fold higher yield of RC 101 than purification by affinity chromatography using His-tag. In planta bioassays with Erwinia carotovora confirmed the antibacterial activity of RC101 and PG1 expressed in chloroplasts. RC101 transplastomic plants were resistant to TMV infections, confirming antiviral activity. Because RC101 and PG1 have not yet been produced in other cell culture or microbial systems, chloroplasts can be used as bioreactors for producing these proteins. Adequate yield of purified antimicrobial peptides from transplastomic plants should facilitate further pre-clinical studies

No description available.

Streptomycin resistance in E. coli ribosomes is conferred by alterations in the amino acid sequence of 30S ribosomal protein S12. The alterations result from point mutations at specific locations in the rps12 gene. A point mutation at a conserved nucleotide in the 16S rRNA gene, originally identified in Euglena gracilis chloroplasts, also confers streptomycin resistance to prokaryotic-like ribosomes. The Nicotiana tabacum mutant "SR1" possesses a chloroplast-linked streptomycin resistance allele. The results presented in this thesis identify a mutation in SR116S rRNA, which occurs at the same position as in streptomycin resistant Euglena mutants. The tobacco chloroplast rps12 gene has been characterized. This gene is expressed in a unique way; two separate transcripts encoding different portions of the gene undergo a bimolecular (trans-) splicing event during mRNA maturation. C-terminal rps12 exons 2 and 3 were identified in the inverted repeat regions of the tobacco chloroplast genome. Complementary DNA sequencing of mature rps12 mRNA allowed deduction of the remaining N-terminal (exon 1) sequence. Hybridizations with synthetic oligodeoxyribonucleotide primers complementary to the deduced RNA sequence located the coding region of exon 1 to be 29 kilobasepairs (kbp) downstream of the nearest copy, and 69 kbp away from, and on the opposite DNA strand of, the distal copy, of exons 2 and 3. Northern hybridization analysis and primer extension sequencing of cDNA of rps12 transcripts indicate that exon 1 and exons 2-3 are encoded on separate transcripts. Exon 1 and exons 2-3 are covalently ligated in mature rps12 mRNA. Therefore, the separate transcripts encoding exon 1 and exons 2-3 undergo a trans-splicing event during the maturation of rps12 mRNA. A complete cloned library of tobacco chloroplast DNA was obtained, consisting of overlapping Bam HI restriction fragments. Three new restriction maps of tobacco chloroplast DNA, for the enzymes Sma I, Kpn I, and Bam HI, were derived by two-dimensional gel analysis and a novel computer-aided mapping technique.

No description available.

<p>As a consequence of the presence of both mitochondria and chloroplasts in plant cells there is a higher sorting requirement in a plant cell than that in a non-plant cell. Reflecting this, protein import to mitochondria and chloroplasts has been shown to be highly specific. However, there is a group of proteins which are encoded by a single gene in the nucleus, translated in the cytosol and targeted to both mitochondria and chloroplasts. These proteins are referred to as dual targeted proteins. The first protein shown to be dual targeted was pea glutathione reductase (GR). The focus of this thesis is the targeting properties of the dual targeted protein glutathione reductase.</p><p>In order to overcome the limitations with traditional in vitro import systems we have developed an import system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The chloroplastic precursor of the small subunit of ribulose bisphosphate carboxylase/oxygenase (SSU) was mis-targeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual system. The dual GR reductase precursor was targeted to both mitochondria and chloroplasts in both the single and dual import system.</p><p>We have investigated the targeting and processing properties of the GR targeting signal. Using N-terminal truncations we have demonstrated that the GR targeting signal has a domain organisation. Our results show that GR has evolved a dual targeting signal with the C-terminal part being sufficient for chloroplast import, the internal part required for the mitochondrial import and the N-terminal part housing a “fine-tuning” function. Furthermore, we have constructed a range of point mutations on the GR signal sequence changing positive amino acid residues and stretches of hydrophobic amino acid residues. Overall single mutations had a greater effect on mitochondrial import compared to import into chloroplasts. We have also shown that the recognition of the GR processing site differs between MPP and SPP. Single amino acid substitutions in the vicinity of the processing site clearly affected processing by MPP while processing by SPP showed low sensitivity to single mutations.</p>

As a consequence of the presence of both mitochondria and chloroplasts in plant cells there is a higher sorting requirement in a plant cell than that in a non-plant cell. Reflecting this, protein import to mitochondria and chloroplasts has been shown to be highly specific. However, there is a group of proteins which are encoded by a single gene in the nucleus, translated in the cytosol and targeted to both mitochondria and chloroplasts. These proteins are referred to as dual targeted proteins. The first protein shown to be dual targeted was pea glutathione reductase (GR). The focus of this thesis is the targeting properties of the dual targeted protein glutathione reductase. In order to overcome the limitations with traditional in vitro import systems we have developed an import system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The chloroplastic precursor of the small subunit of ribulose bisphosphate carboxylase/oxygenase (SSU) was mis-targeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual system. The dual GR reductase precursor was targeted to both mitochondria and chloroplasts in both the single and dual import system. We have investigated the targeting and processing properties of the GR targeting signal. Using N-terminal truncations we have demonstrated that the GR targeting signal has a domain organisation. Our results show that GR has evolved a dual targeting signal with the C-terminal part being sufficient for chloroplast import, the internal part required for the mitochondrial import and the N-terminal part housing a “fine-tuning” function. Furthermore, we have constructed a range of point mutations on the GR signal sequence changing positive amino acid residues and stretches of hydrophobic amino acid residues. Overall single mutations had a greater effect on mitochondrial import compared to import into chloroplasts. We have also shown that the recognition of the GR processing site differs between MPP and SPP. Single amino acid substitutions in the vicinity of the processing site clearly affected processing by MPP while processing by SPP showed low sensitivity to single mutations.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 110-137). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Oregon, 2000. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 56-59). Also available for download via the World Wide Web; free to University of Oregon users.

Immunocytochemical analyses have demonstrated that the Calvin cycle enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is predominantly localized in the pyrenoid region of chloroplasts of evolutionarily diverse algae. That Rubisco remains pyrenoid-localized at photosynthetically-saturating irradiance in the green alga Chlorella pyrenoidosa indicates a catalytic, rather than storage function for pyrenoid-localized Rubisco. This is further supported by the immunolocalization of Rubisco activase to the pyrenoids of two species of green algae. The exclusion of phosphoribulokinase from the pyrenoids of a red and a green alga indicates that pyrenoids do not possess the full complement of Calvin cycle enzymes. / Thylakoid lamellae traverse the pyrenoids of many algae. The absence of light-harvesting phycoerythrin and of photosystem (PS) II activity, but not PSI activity, from the intrapyrenoid thylakoids of the red alga Porphyridium cruentum indicates a structural and functional heterogeneity between these lamellae and those located in the chloroplast stroma. In contrast, the intrapyrenoid thylakoids of cryptomonads, algae whose chloroplast is thought to have evolved from red algae, possess both PSI and PSII protein complexes. These results are discussed with reference to Rubisco being mainly pyrenoid-localized in these algae.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 3 uppsatser.