Chloroplast and cytoplasmic ribosomes have been isolated from a number of species of the angiosperm genus Nicotiana. Ribosomal subunit and monosome proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Resultant two-dimensional electrophoretic patterns of chloroplast and cytoplasmic ribosomal proteins were processed by a computer algorithm, developed to formally compare different electrophoretic patterns by the construction of two-dimensional, conformal average electrophoretic mobility maps. The chloroplast ribosomal subunit of N. tabacum contains 22-24 distinct basic polypeptides (pI > 5) and 2-3 acidic proteins (pI < 5). The 50S chloroplast ribosomal subunit possesses at least 1 acidic and 33-35 basic proteins. 40S and 60S cytoplasmic ribosomal subunits of the same species have 26-30 and 47-50 basic polypeptides, respectively. Molecular weights of chloroplast ribosomal proteins (ChRP) and cytoplasmic ribosomal proteins (CyRP) were estimated. There was little similarity between the 2D electrophoretic patterns of ChRP and CyRP of N. tabacum. However, 2D-PAGE patterns of N. tabacum ChRP and CyRP were qualitatively isomorphous with homologous patterns of Chlamydomonas reinhardi, pea and spinach. In terms of molecular weight and electrophoretic pattern N. tabacum ChRP were found to be more closely affiliated with prokaryotic ribosomal proteins than with CyRP from the same species. ChRP were isolated from N. gossei (an Australian species) and its reciprocol interspecies hybrids with N. tabacum (denoted by: T x G and G x T). Interspecies polymorphisms between homologous N. tabacum and N. gossei ChRP were delineated by computerized mobility mapping and co-electrophoresis of radiolabeled N. tabacum ChRP with a large molar excess of N. gossei ChRP. The inheritance mode (Mendelian vs. maternal) of a number of well-defined interspecies ChRP polymorphisms was determined by co-electrophoresis of radioiodinated N. tabacum ChRP with T x G and G x T hybrid ChRP. Results indicate that at least 4 30S ChRP and 3 50S ChRP are encoded by nuclear genes. 30S ChRP from an N. tabacum line carrying a maternally-inherited streptomycin-resistance mutation (SR-1) were compared to N. tabacum 30S ChRP by mobility mapping. Two differences were established between the SR-1 and wild-type 30S ChRP average mobility maps. These findings correlate with the reduced affinity of SR-1 30S chloroplast ribosomal subunits for ('3)H-dihydrostreptomycin, and show that at least one 30S ChRP is encoded by chloroplast DNA. Preparative 2D-PAGE and reverse high performance liquid chromatography (RPHPLC) separation techniques for complex ribosomal protein mixtures were developed. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI

The structure and expression of a novel Euglena gracilis chloroplast ribosomal protein operon was studied by gene mapping, molecular cloning, nucleotide sequencing primer extension and Northern analyses. The nucleotide sequence (12,240 bp) was determined for 100% of both strands encoding the 12 genes, rpl23 - rpl2 - rps19 - rpl22 - rps3-(2.8 kb region)- rpl16 - rpl14 - rpl5 - rps8 - rpl36 - trnI - rps14. The gene organization resembles the S10 and spc ribosomal protein operons of E. coli. The rpl5 gene was a new chloroplast gene not previously reported for any chloroplast genome nor described as a nuclear gene. The presence of numerous introns and an unusual 2.8 kb rps3-rpl16 intercistronic region were additional features that were unparalleled in other chloroplast DNAs. At least 15 introns were identified in the genes. Evidence is presented from primer extension analysis of chloroplast RNA for the correct in vivo splicing of six of the introns. Two introns within rps8 flanked an 8 bp exon, the smallest exon yet characterized in a chloroplast genome. Four introns shared structural properties with group II organelle introns. The remaining 11 introns were defined as new category of organelle intron, now designated "group III." The presence of additional introns in several intercistronic regions is proposed. Conserved regions in the predicted polypeptides were identified from the alignments with related proteins from other chloroplasts and bacteria. Evidence from Northern hybridization experiments with gene-specific probes supported the interpretation that 11 ribosomal protein genes, the 2.8 kb rps3-rpl16 intercistronic region and trnI were co-transcribed and encoded in a single operon. The co-transcription of genes coding for proteins and a tRNA is a novel finding for a chloroplast operon. Several stable polycistronic transcripts were identified, including a common 8.3 kb pre-mRNA. Stepwise processing pathways proposed for the mRNAs are described. Most mRNAs appeared to be fully spliced. The 5$\sp\prime$ ends of mRNAs for the first gene in the operon, rpl23, were mapped by primer extension. Plastid mRNAs from dark and light grown Euglena were analyzed on Northern blots.

Proteínas FtsH são metaloproteases pertencentes à família AAA (ATPases Associadas a Diversas Atividades Celulares), e estão presentes em todos os reinos dos seres vivos. Estas proteases utilizam a energia liberada da hidrólise do ATP para desempenhar suas diversas atividades celulares. Em Escherichia coli, as proteases FtsH se organizam em um homohexamero na membrana plasmática, sendo que este complexo atua na degradação de proteínas mal dobradas. Em Arabidopsis, o hetero-complexo FtsH localizado na membrana dos tilacóides é formados por isômeros do tipo A (FtsH1/FtsH5) e do tipo B (FtsH2/FtsH8). Sua atividade proteolítica está relacionada ao controle de qualidade organelar, degradando proteínas mal dobradas e de vida curta. O complexo está envolvido também na degradação da proteína D1 do PSII, danificada por danos foto-oxidativo. Embora as FtsH cloroplastidiais sejam bem caracterizadas em termos genéticos e moleculares, o mecanismo de regulação do complexo ainda não foi esclarecido, o que torna interessante a busca por fatores proteicos adicionais. Em investigações anteriores nosso grupo de pesquisa encontrou um candidato potencial, denominado FIP (FtsH5 Interacting Protein), que pode modular a atividade ATPásica e/ou proteásica do complexo. Fip está presente na membrana dos tilacóides e mostrou interação com FtsH5 in vivo e in vitro. Neste trabalho foram realizados ensaios de duplo híbrido de leveduras, utilizando deleções da proteína FIP, bem como substituições de resíduos de cisteína por alanina. Os resultados revelaram que a interação entre FIP e FtsH5 é mantida somente quando duas regiões ricas em cisteína estão presentes na sequência de FIP. Essa região compreende 46 aminoácidos, com 4 resíduos de cisteína conservados, e ensaios com 7 diferentes substituições desses resíduos por alanina não mostraram interação com FtsH5, o que corrobora a hipótese de que os resíduos de cisteína são necessários para a interação. Experimentos de análise de expressão utilizando PCR em tempo real, sob condições de estresse salino e estresse a frio, revelaram que os genes que codificam FIP e FtsH5 têm sua expressão regulada de modo antagônico, o que sugere que FIP possa atuar como modulador negativo da atividade proteásica do complexo FtsH. / Metalloprotease FtsH proteins are members of the AAA family (ATPases Associated with Diverse Cellular Activities), and are present in all kingdoms of living organisms. These proteases use energy of ATP hydrolysis to perform its various cellular activities. In Escherichia coli, FtsH proteases are organized in a homo-hexamer in the cytoplasmic membrane, and this complex acts in the degradation of misfolded proteins. In Arabidopsis, the FtsH hetero-complex located in the thylakoid membrane is formed by type A (FtsH1/FtsH5) and type B (FtsH2/FtsH8) isomers. Its proteolytic activity is involved in organellar quality control by degrading misfolded and short-lived proteins. The complex is also involved in the degradation of the D1 protein of PSII, damaged by photo-oxidative damage. Although the chloroplast FtsH are well characterized genetically and molecularly, the regulatory mechanism of the complex remains unclear, which makes it interesting to search for additional protein factors. In earlier studies our research group has found a potential candidate called FIP (FtsH5 Interacting Protein), which can modulate the ATPase and/or protease activity of the complex. FIP is present in the membrane of the thylakoids and showed interaction with FtsH5 in vivo and in vitro. In this study yeast two-hybrid assays were performed using FIP protein deletions, and substitutions of cysteine to alanine residues. The results showed that the interaction between FIP and FtsH5 is maintained only when two cysteine rich regions are present in the sequence of FIP. This region contains 46 amino acids with four conserved cysteine residues and 7 different assays with alanine replacements of these residues showed no interaction with FtsH5, which corroborates the hypothesis that the cysteine residues are required for the interaction. Expression experiments analysis using realtime PCR, under conditions of salt stress and cold stress, revealed that the genes encoding FtsH5 and FIP have its expression regulated in an antagonistic way, suggesting that FIP can act as a negative modulator of the activity FtsH protease of the complex.

Chloroplast genomes of land plants show conservation of structure and gene arrangement. The spinach chloroplast genome is comprised of a covalently closed. circular DNA molecule of 150 kilobases and is typical of these plants. Approximately 20% of the proteins found in the spinach chloroplast are encoded by the chloroplast genome and translated on chloroplast ribosomes. The remainder are encoded on chromosomes in the nucleus, translated on cytoplasmic ribosomes and transported into the chloroplast. Spinach chloroplast DNA was isolated from crude 2 chloroplast preparations by a new method. Chloroplasts were lysed with alkaline sodium dodecyl sulfate, contaminating macromolecules precipitated with acidified potassium acetate and plastid DNA was purified by phenol:chloroform extraction and ethanol:ammonium acetate precipitation. The yield was approximately 50 ug chloroplast DNA per 100 grams leaf material. The DNA consisted of 10% circular molecules and 90% linear molecules. The chloroplast DNA was digested with restriction enzyme PstI and the fragments were cloned into the plasmid vector pUC9. Several recombinant plasmids were isolated and the chloroplast DNA inserts identified. The recombinant plasmid pRD105 containing the PstI #5 fragment was subjected to further investigation. The ClaI restriction sites of the PstI #5 fragment were mapped and the insert was subcloned into the plasmid vector pGEM4, which bears bacteriophage SP6 and T7 RNA polymerase promoter sequences.

<p>The information for mitochondrial and chloroplastic protein import and targeting generally resides in the N-terminal part of the protein, called a targeting peptide. The targeting peptide is cleaved off by the organellar processing peptidases after import of a precursor protein. Free targeting peptides generated inside the organelle after import are rapidly degraded by proteolysis as their accumulation can have toxic effects on the organelle. The aim of this thesis has been to investigate the newly identified targeting peptide degrading protease, the PresequenceProtease (PreP). We have shown that the two isoforms of <i>Arabidopsis </i>PreP (<i>At</i>PreP1 and <i>At</i>PreP1) are dually targeted and localized to both mitochondria and chloroplasts. Dual targeting of the <i>At</i>PreP1 is due to an ambiguous targeting peptide with a domain organization for mitochondrial and chloroplastic targeting. Both the <i>At</i>PreP1 and <i>At</i>PreP2 are expressed in <i>Arabidopsis</i> in an organ specific manner and they have distinct but overlapping substrate specificity. The crystal structure of the recombinant <i>At</i>PreP1 E80Q was solved at 2.1 Å resolution. The structure represents the first substrate bound, closed conformation of a protease from the pitrilysin family. The PreP polypeptide folds in a unique peptidasome structure, surrounding a huge cavity of more than 10 000 Å3 in which the active site resides. A novel mechanism for proteolysis is proposed involving hinge-bending motions in response to substrate binding. PreP in human mitochondria has a novel function: degradation of amyloid β-peptide (Aβ). We show that under circumstances when Aβ is present in mitochondria of Alzheimer’s patients, PreP is the protease responsible for degradation of this toxic peptide. </p>

The information for mitochondrial and chloroplastic protein import and targeting generally resides in the N-terminal part of the protein, called a targeting peptide. The targeting peptide is cleaved off by the organellar processing peptidases after import of a precursor protein. Free targeting peptides generated inside the organelle after import are rapidly degraded by proteolysis as their accumulation can have toxic effects on the organelle. The aim of this thesis has been to investigate the newly identified targeting peptide degrading protease, the PresequenceProtease (PreP). We have shown that the two isoforms of Arabidopsis PreP (AtPreP1 and AtPreP1) are dually targeted and localized to both mitochondria and chloroplasts. Dual targeting of the AtPreP1 is due to an ambiguous targeting peptide with a domain organization for mitochondrial and chloroplastic targeting. Both the AtPreP1 and AtPreP2 are expressed in Arabidopsis in an organ specific manner and they have distinct but overlapping substrate specificity. The crystal structure of the recombinant AtPreP1 E80Q was solved at 2.1 Å resolution. The structure represents the first substrate bound, closed conformation of a protease from the pitrilysin family. The PreP polypeptide folds in a unique peptidasome structure, surrounding a huge cavity of more than 10 000 Å3 in which the active site resides. A novel mechanism for proteolysis is proposed involving hinge-bending motions in response to substrate binding. PreP in human mitochondria has a novel function: degradation of amyloid β-peptide (Aβ). We show that under circumstances when Aβ is present in mitochondria of Alzheimer’s patients, PreP is the protease responsible for degradation of this toxic peptide.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 19, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Thesis advisor: Dr. James E. Schoelz. Includes bibliographical references.

No description available.

Members of the genus Packera (Asteraceae) are widespread in North America, but most are found in western regions of the continent where extensive morphological integradation is common. Previous molecular systematic studies found that four species in southwestern Alberta, a region proposed to be at the interface of the Cordilleran and Laurentide ice sheets during the last advance of Pleistocene glaciation, showed unusually high levels of inter- and intrapopulational chloroplast DNA variation. The present study analyzed chloroplast haplotype phylogeny, frequency variation, and geographic distribution patterns in Packera contermina and closely related species P. pseudaurea, P. cana, and P. cymbalarioides from southwestern Alberta, nothern Montana, and northwestern Wyoming. Restriction site analyses of chloroplast DNA from 730 individuals across 34 populations of the four species revealed fifteen haplotypes, of which seven are commonly found in other North American Packera species. Three haplotypes were detected in P. cymbalarioides, seven in P. cana, eight in P. pseudaura, and twelve in P. contermina. The level of ahplotype frequency variation among populations was high in P. cymbalarioides (0=I) moderate to low in P. contermina (0=0.333) and P.cana (0=0.261), and very low in P.pseudaurea (0=0.085), possibly reflecting differences in the species' history. Phylogenetic analyses revealed 2 groups of haplotypes, one of which is found mainly in populations from the Great Basin of North America and the second in populations of more coastal and northern regions. The presence of haplotypes from both groups of Packera species suggests that the cpDNA diversity in southern Alberta arisen through hybridization/introgression events that have involved a number of species from outside of the region. / x, 81 leaves : ill., maps ; 28 cm.

The technique of restriction fragment length polymorphism (RFLP) analysis, of chloroplastic and genomic DNA, was investigated as a means of identifying eucalypt species and cultivars which are morphologically indistinguishable from one another. In order to resolve chloroplast DNA (cpDNA) RFLPs, a method was developed to extract high yields of intact chloroplasts from Eucalyptus grandis S/N M6. Starch contamination was reduced by incubation of saplings in the dark for 48 h prior to extraction and watering with a solution containing 370 mM Na-phosphate and 296 mM KN03. Optimal chloroplast yields (25 ug chlorophyll/g fresh mass) were obtained by chopping leaf material, using a vertical homogenizer, in a buffer containing 350 mM sorbitol, 50 mM tris-HCL and 5 mM EDTA, 0.1 % (w/v) bovine serum albumin, 0.15 % (w/v) 2-mercaptoethanol, 2 mM L-ascorbic acid and 1 mM MgCI2 followed by washing of leaf pieces in a buffer containing only sorbitol, tris-HCL and EDTA. When these chloroplasts were used in an "in-organelle" DNA digestion procedure, polymorphisms were observed between the cpDNA profiles resolved for E. grandis S/N M6 and that of an outgroup species (spinach). However, the developed chloroplast extraction technique could not be used to obtain chloroplasts from various other eucalypt species, probably as a result of variability in the material at an ultrastructural or biochemical level. For the analysis of genomic DNA RFLPs, a DNA extraction procedure was optimized for use with various eucalypt species and cultivars. This included the development of a purifcation technique during which DNA was ammonium acetate-ethanol precipitated and subjected to mini-dialysis. Following Dra I restriction of DNA, the extract was electrophoresed and Southern blotted onto both nylon and nitrocellulose membranes. These were probed with a Hind-III restricted sample of the multilocus plasmid probe pV47-2. This probe was labelled using 32p as well as a non-radioactive labelling substance digoxygenin (DIG). Hybridization conditions, including the composition of the hybridization buffer, were optimized for use with these labels, and DNA RFLPs (fingerprints) were resolved for the eucalypt species E. grandis and E. macarthurii and cultivars of E. grandis (S/N M6, TAG 5 and TAG 14). An average of 8.5 bands were detected with 32p and 5.0 fragments with DIG. All the species and cultivars fingerprinted with the 32P-label could be distinguished from one another. However, as a result of the reduced sensitivity of the DIG system, two of the E. grandis cultivars, S/N M6 and TAG 5, could not be differentiated. It is concluded that the latter system would be most suitable for incorporation into a routine eucalypt screening programme, although it is suggested that the colourimetric detection assay, used in this study to resolve DNA bands, be replaced by a more sensitive one. / Thesis (M.Sc)-University of Natal, Durban, 1993.