3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process

1993 ◽  
Vol 13 (4) ◽  
pp. 2277-2285
Author(s):  
D B Stern ◽  
K L Kindle
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Transcription Termination ◽  
Protein Coding ◽  
Coding Regions ◽  
In Vitro Processing ◽  
Mrna Precursor ◽  
Chloroplast Transcripts

Inverted repeat (IR) sequences are found at the 3' ends of most chloroplast protein coding regions, and we have previously shown that the 3'IR is important for accumulation of atpB mRNA in Chlamydomonas reinhardtii (D. B. Stern, E.R. Radwanski, and K. L. Kindle, Plant Cell 3:285-297, 1991). In vitro studies indicate that 3' IRs are inefficient transcription termination signals in higher plants and have furthermore defined processing activities that act on the 3' ends of chloroplast transcripts, suggesting that most chloroplast mRNAs are processed at their 3' ends in vivo. To investigate the mechanism of 3' end processing in Chlamydomonas reinhardtii chloroplasts, the maturation of atpB mRNA was examined in vitro and in vivo. In vitro, a synthetic atpB mRNA precursor is rapidly cleaved at a position 10 nucleotides downstream from the mature 3' terminus. This cleavage is followed by exonucleolytic processing to generate the mature 3' end. In vivo run-on transcription experiments indicate that a maximum of 50% of atpB transcripts are transcriptionally terminated at or near the IR, while the remainder are subject to 3' end processing. Analysis of transcripts derived from chimeric atpB genes introduced into Chlamydomonas chloroplasts by biolistic transformation suggests that in vivo processing and in vitro processing occur by similar or identical mechanisms.

scholarly journals 3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process.

1993 ◽  
Vol 13 (4) ◽  
pp. 2277-2285 ◽  
Author(s):  
D B Stern ◽  
K L Kindle
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Transcription Termination ◽  
Protein Coding ◽  
Coding Regions ◽  
In Vitro Processing ◽  
Mrna Precursor ◽  
Chloroplast Transcripts

Inverted repeat (IR) sequences are found at the 3' ends of most chloroplast protein coding regions, and we have previously shown that the 3'IR is important for accumulation of atpB mRNA in Chlamydomonas reinhardtii (D. B. Stern, E.R. Radwanski, and K. L. Kindle, Plant Cell 3:285-297, 1991). In vitro studies indicate that 3' IRs are inefficient transcription termination signals in higher plants and have furthermore defined processing activities that act on the 3' ends of chloroplast transcripts, suggesting that most chloroplast mRNAs are processed at their 3' ends in vivo. To investigate the mechanism of 3' end processing in Chlamydomonas reinhardtii chloroplasts, the maturation of atpB mRNA was examined in vitro and in vivo. In vitro, a synthetic atpB mRNA precursor is rapidly cleaved at a position 10 nucleotides downstream from the mature 3' terminus. This cleavage is followed by exonucleolytic processing to generate the mature 3' end. In vivo run-on transcription experiments indicate that a maximum of 50% of atpB transcripts are transcriptionally terminated at or near the IR, while the remainder are subject to 3' end processing. Analysis of transcripts derived from chimeric atpB genes introduced into Chlamydomonas chloroplasts by biolistic transformation suggests that in vivo processing and in vitro processing occur by similar or identical mechanisms.

Structural and functional consequences of the replacement of proximal residues Cys172 and Cys192 in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

2008 ◽  
Vol 411 (2) ◽  
pp. 241-247 ◽  
Author(s):  
María-Jesús García-Murria ◽  
Saeid Karkehabadi ◽  
Julia Marín-Navarro ◽  
Sriram Satagopan ◽  
Inger Andersson ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Large Subunit ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Bisphosphate Carboxylase ◽  
Specificity Factor

Proximal Cys172 and Cys192 in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) are evolutionarily conserved among cyanobacteria, algae and higher plants. Mutation of Cys172 has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here, we report the effect of the replacement of Cys172 and Cys192 by serine on the catalytic properties, thermostability and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor when compared with the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The Vc (Vmax for carboxylation) was similar to that of wild-type Rubisco in the case of the C172S enzyme, but approx. 30% lower for the C192S Rubisco. In contrast, the Km for CO2 and O2 was similar for C192S and wild-type enzymes, but distinctly higher (approximately double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature approx. 2 °C lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 °C, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main-chain backbone atoms of β-strand 1 of the α/β-barrel affecting a number of amino acid side chains. This may cause the exceptional catalytic features of C172S. In contrast, the C192S mutation does not produce similar structural perturbations.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

scholarly journals In Vitro Import of a Nuclearly Encoded tRNA into Mitochondria of Solanum tuberosum

2003 ◽  
Vol 23 (11) ◽  
pp. 4000-4012 ◽  
Author(s):  
Ludovic Delage ◽  
André Dietrich ◽  
Anne Cosset ◽  
Laurence Maréchal-Drouard
Keyword(s):  
Solanum Tuberosum ◽  
Higher Plants ◽  
Plant Mitochondria ◽  
Protein Translation ◽  
Trna Genes ◽  
Vitro Assay ◽  
Trna Import

ABSTRACT Some of the mitochondrial tRNAs of higher plants are nuclearly encoded and imported into mitochondria. The import of tRNAs encoded in the nucleus has been shown to be essential for proper protein translation within mitochondria of a variety of organisms. Here, we report the development of an in vitro assay for import of nuclearly encoded tRNAs into plant mitochondria. This in vitro system utilizes isolated mitochondria from Solanum tuberosum and synthetic tRNAs transcribed from cloned nuclear tRNA genes. Although incubation of radioactively labeled in vitro-transcribed tRNAAla, tRNAPhe, and tRNAMet-e with isolated potato mitochondria resulted in importation, as measured by nuclease protection, the amount of tRNA transcripts protected at saturation was at least five times higher for tRNAAla than for the two other tRNAs. This difference in in vitro saturation levels of import is consistent with the in vivo localization of these tRNAs, since cytosolic tRNAAla is naturally imported into potato mitochondria whereas tRNAPhe and tRNAMet-e are not. Characterization of in vitro tRNA import requirements indicates that mitochondrial tRNA import proceeds in the absence of any added cytosolic protein fraction, involves at least one protein component on the surface of mitochondria, and requires ATP-dependent step(s) and a membrane potential.

scholarly journals In vitro processing at the 3'-terminal region of pre-18S rRNA by a nucleolar endoribonuclease.

1990 ◽  
Vol 10 (8) ◽  
pp. 3868-3872 ◽  
Author(s):  
C M Shumard ◽  
C Torres ◽  
D C Eichler
Keyword(s):  
18S Rrna ◽  
Precise Determination ◽  
In Vivo Studies ◽  
Rrna Sequence ◽  
S1 Nuclease ◽  
In Vitro Processing ◽  
Rrna Maturation ◽  
A Site

In an investigation of the possible involvement of a highly purified nucleolar endoribonuclease in processing of pre-rRNA at the 3' end of the 18S rRNA sequence, an in vitro synthesized pre-18S rRNA transcript containing the 3' end region of 18S rRNA and the 5' region of the first internal transcribed spacer (ITS1) was used as a substrate for the enzyme. Cleavages generated by the nucleolar RNase were localized by S1 nuclease protection analysis and by the direct release of labeled rRNA products. Precise determination of the specificity of cleavage was achieved by RNA sequence analysis with end-labeled rRNA transcripts. These data demonstrated that the purified nucleolar RNase cleaved the pre-18S rRNA transcript at three specific sites relative to the 3' region of 18S rRNA. The first two sites included the mature 3'-end 18S rRNA sequence and a site approximately 55 nucleotides downstream of the 3'-end 18S rRNA sequence, both of which corresponded directly to recent results (Raziuddin, R. D. Little, T. Labella, and D. Schlessinger, Mol. Cell. Biol. 9:1667-1671, 1989) obtained with transfected mouse rDNA in hamster cells. The other cleavage occurred approximately 35 nucleotides upstream from the mature 3' end in the 18S rRNA sequence. The results from this study mimic the results obtained from in vivo studies for processing in the 3' region of pre-18S rRNA, supporting the proposed involvement of this nucleolar endoribonuclease in rRNA maturation.

scholarly journals A Review of Plants Used in South African Traditional Medicine for the Management and Treatment of Hypertension

Planta Medica ◽  
2018 ◽  
Vol 85 (04) ◽  
pp. 312-334 ◽  
Author(s):  
Fatai Balogun ◽  
Anofi Ashafa
Keyword(s):  
South Africa ◽  
Medicinal Plants ◽  
South African ◽  
Plant Species ◽  
Agricultural Research ◽  
Higher Plants ◽  
In Vivo Studies ◽  
Research Councils

AbstractSouth Africa contains 9% of the worldʼs higher plants, and despite its rich biodiversity, it has one of the highest prevalence of hypertension in Africa. This review provides information on medicinal plants embraced in South Africa for hypertension management, with the aim of reporting pharmacological information on the indigenous use of these plants as antihypertensives. This review not only focuses on the activity of antihypertensive medicinal plants but also reports some of its phytochemical constituents and other ethnopharmacological and therapeutic properties. Information obtained from scientific and or unpublished databases such as Science Direct, PubMed, SciFinder, JSTOR, Google Scholar, Web of Science, and various books revealed 117 documented antihypertensive plant species from 50 families. Interestingly, Asteraceae topped the list with 16 species, followed by Fabaceae with 8 species; however, only 25% of all plant species have demonstrated antihypertensive effects originating from both in vitro and in vivo studies, lending credence to their folkloric use. Only 11 plant species reportedly possess antihypertensive properties in animal models, with very few species subjected to analytical processes to reveal the identity of their bioactive antihypertensive compounds. In this review, we hope to encourage researchers and global research institutions (universities, agricultural research councils, and medical research councils), particularly those showing an interest in natural products, for the need for concerted efforts to undertake more studies aimed at revealing the untapped potential of these plants. These studies are very important for the development of new pharmaceuticals of natural origin useful for the management of hypertension.

Overexpression of a plant cysteine synthase gene and biosynthesis of a plant specific metabolite, β-(pyrazol-1-yl)-L-alanine, in Escherichia coli

1994 ◽  
Vol 72 (1) ◽  
pp. 188-192 ◽  
Author(s):  
Kazuki Saito ◽  
Reiko Kanda ◽  
Makoto Kurosawa ◽  
Isamu Murakoshi
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Control ◽  
Spinacia Oleracea ◽  
Higher Plants ◽  
Total Soluble Protein ◽  
T7 Promoter ◽  
Cysteine Synthase ◽  
Mechanistic Investigation

Cysteine synthase (EC 4.2.99.8) in higher plants is responsible for biosynthesis of not only cysteine but also some nonprotein amino acids such as β-(pyrazol-1-yl)-L-alanine. The cDNA of a cysteine synthase from spinach (Spinacia oleracea) was inserted into pET8c (=pET3d) under the transcriptional control of strong T7 promoter to yield an overexpression vector pCEK1. The amount of the exogenous cysteine synthase was increased up to 40% of the total soluble protein of Escherichia coli transformed with pCEK1. β-(Pyrazol-1-yl)-L-alanine, a specific metabolite in plants of the Cucurbitaceae, was biosynthesized by overexpressed cysteine synthase from pyrazole in the presence of O-acetyl-L-serine and serine, in vitro and in vivo, respectively. The present study provides the system for mechanistic investigation of biosynthesis of cysteine and biogenetically related β-substituted alanines at molecular genetic level.

scholarly journals Identification of parallel pH- and zeaxanthin-dependent quenching of excess energy in LHCSR3 in Chlamydomonas reinhardtii

2020 ◽  
Author(s):  
Julianne M. Troiano ◽  
Federico Perozeni ◽  
Raymundo Moya ◽  
Luca Zuliani ◽  
Kwangryul Baek ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Light Harvesting ◽  
Complex Stress ◽  
Excess Energy ◽  
Photochemical Quenching ◽  
Light Harvesting Complexes ◽  
Light Harvesting Complex ◽  
Non Photochemical Quenching

AbstractUnder high light conditions, oxygenic photosynthetic organisms avoid photodamage by thermally dissipating excess absorbed energy, which is called non-photochemical quenching (NPQ). In green algae, a chlorophyll and carotenoid-binding protein, light-harvesting complex stress-related (LHCSR3), detects excess energy via pH and serves as a quenching site. However, the mechanisms by which LHCSR3 functions have not been determined. Using a combined in vivo and in vitro approach, we identify two parallel yet distinct quenching processes, individually controlled by pH and carotenoid composition, and their likely molecular origin within LHCSR3 from Chlamydomonas reinhardtii. The pH-controlled quenching is removed within a mutant LHCSR3 that lacks the protonable residues responsible for sensing pH. Constitutive quenching in zeaxanthin-enriched systems demonstrates zeaxanthin-controlled quenching, which may be shared with other light-harvesting complexes. We show that both quenching processes prevent the formation of damaging reactive oxygen species, and thus provide distinct timescales and mechanisms of protection in a changing environment.

scholarly journals MicroRNA-1205 Regulation of FRYL in Prostate Cancer

2021 ◽  
Vol 9 ◽  
Author(s):  
Michelle Naidoo ◽  
Fayola Levine ◽  
Tamara Gillot ◽  
Akintunde T. Orunmuyi ◽  
E. Oluwabunmi Olapade-Olaopa ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Neuroendocrine Differentiation ◽  
Castration Resistant Prostate Cancer ◽  
Chromosomal Locus ◽  
Protein Coding ◽  
Castration Resistant ◽  
Dendritic Branching ◽  
Very High

High mortality rates of prostate cancer (PCa) are associated with metastatic castration-resistant prostate cancer (CRPC) due to the maintenance of androgen receptor (AR) signaling despite androgen deprivation therapies (ADTs). The 8q24 chromosomal locus is a region of very high PCa susceptibility that carries genetic variants associated with high risk of PCa incidence. This region also carries frequent amplifications of the PVT1 gene, a non-protein coding gene that encodes a cluster of microRNAs including, microRNA-1205 (miR-1205), which are largely understudied. Herein, we demonstrate that miR-1205 is underexpressed in PCa cells and tissues and suppresses CRPC tumors in vivo. To characterize the molecular pathway, we identified and validated fry-like (FRYL) as a direct molecular target of miR-1205 and observed its overexpression in PCa cells and tissues. FRYL is predicted to regulate dendritic branching, which led to the investigation of FRYL in neuroendocrine PCa (NEPC). Resistance toward ADT leads to the progression of treatment related NEPC often characterized by PCa neuroendocrine differentiation (NED), however, this mechanism is poorly understood. Underexpression of miR-1205 is observed when NED is induced in vitro and inhibition of miR-1205 leads to increased expression of NED markers. However, while FRYL is overexpressed during NED, FRYL knockdown did not reduce NED, therefore revealing that miR-1205 induces NED independently of FRYL.

scholarly journals The closely related Drosophila sry beta and sry delta zinc finger proteins show differential embryonic expression and distinct patterns of binding sites on polytene chromosomes

Development ◽  
1990 ◽  
Vol 110 (1) ◽  
pp. 141-149 ◽  
Author(s):  
F. Payre ◽  
S. Noselli ◽  
V. Lefrere ◽  
A. Vincent
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Polytene Chromosomes ◽  
Duplication Event ◽  
Amino Acid Sequences ◽  
Evolutionary Divergence ◽  
Coding Region ◽  
Protein Coding

Serendipity (sry) beta (beta) and delta (delta) are two finger protein genes resulting from a duplication event. Comparison of their respective protein products shows interspersed blocks of conserved and divergent amino-acid sequences. The most extensively conserved region corresponds to the predicted DNA-binding domain which includes 6 contiguous fingers; no significant sequence conservation is found upstream and downstream of the protein-coding region. We have analysed the evolutionary divergence of the sry beta and delta proteins on two separate levels, their embryonic pattern of expression and their DNA-binding properties in vitro and in vivo. By using specific antibodies and transformant lines containing beta-galactosidase fusion genes, we show that the sry beta and sry delta proteins are maternally inherited and present in embryonic nuclei at the onset of zygotic transcription, suggesting that they are transcription factors involved in this process. Zygotic synthesis of the sry beta protein starts during nuclear division cycles 12–13, prior to cellularisation of the blastoderm, while the zygotic sry delta protein is not detectable before germ band extension (stage 10 embryos). Contrary to sry delta, the zygotic sry beta protein constitutes only a minor fraction of the total embryonic protein. The sry beta and delta proteins made in E. coli bind to DNA, with partly overlapping specificities. Their in vivo patterns of binding to DNA, visualised by immunostaining polytene chromosomes, differ both in the number and position of their binding sites. Thus changes in expression pattern and DNA-binding specificity have contributed to the evolution of the sry beta and delta genes.

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