cDNA cloning and gene expression of carbonic anhydrase in Chlamydomonas reinhardtii

1991 ◽  
Vol 69 (5) ◽  
pp. 1088-1096 ◽  
Author(s):  
Hideya Fukuzawa ◽  
Sarami Ishida ◽  
Shigetoh Miyachi
Keyword(s):  
Carbonic Anhydrase ◽  
Chlamydomonas Reinhardtii ◽  
Sequence Similarity ◽  
Large Subunit ◽  
Mrna Level ◽  
Nuclear Genome ◽  
Small Subunit ◽  
Nucleotide Sequence Analysis ◽  
Cdna Clones ◽  
Upstream Gene

cDNA and genes encoding periplasmic carbonic anhydrase (CA) polypeptides of Chlamydomonas reinhardtii have been isolated and characterized. Nucleotide sequence analysis of cDNA clones revealed that the large subunit (35 kDa or 36.5 kDa) and the small subunit (4 kDa) are cotranslated as a precursor polypeptide (41 626 Da) with a NH2-terminal hydrophobic signal peptide of 20 amino acids. The amino acid sequence of Chlamydomonas CA showed 20–22% identity with animal CA isozymes (CAI, CAII, CAIII, and CAVII). Three zinc-liganded histidine residues and those forming the hydrogen-bond network to zinc-bound solvent molecules were highly conserved. No significant sequence similarity was observed between Chlamydomonas CA and chloroplast CAs of spinach and pea. Two copies of structurally related CA genes (CAH1 and CAH2) were tandemly clustered in Chlamydomonas nuclear genome and regulated by external CO2 concentration in a reverse manner. The 5′ upstream gene CAH1 encodes the major periplasmic CA whose mRNA level is induced under low CO2 condition in light. Photosynthesis is absolutely required for the accumulation of the CAH1 mRNA. The 3′ downstream gene CAH2 is possibly a gene for another periplasmic CA isozyme, which is induced under high CO2 conditions. Light has an inhibitory effect on the accumulation of the CAH2 mRNA. Key words: photosynthesis, light regulation, zinc, CO2-concentrating mechanism, intracellular processing.

scholarly journals Ciliate Environmental Diversity Can Be Underestimated by the V4 Region of SSU rDNA: Insights from Species Delimitation and Multilocus Phylogeny of Pseudokeronopsis (Ciliophora, Spirotrichea)

2019 ◽  
Vol 7 (11) ◽  
pp. 493 ◽  
Author(s):  
Zhan ◽  
Li ◽  
Xu
Keyword(s):  
Species Delimitation ◽  
High Throughput Sequencing ◽  
Sequence Similarity ◽  
Large Subunit ◽  
Small Subunit ◽  
Base Change ◽  
Published Data ◽  
Environmental Diversity ◽  
5.8S Rdna ◽  
Eukaryotic Microorganisms

Metabarcoding and high-throughput sequencing methods have greatly improved our understanding of protist diversity. Although the V4 region of small subunit ribosomal DNA (SSU-V4 rDNA) is the most widely used marker in DNA metabarcoding of eukaryotic microorganisms, doubts have recently been raised about its suitability. Here, using the widely distributed ciliate genus Pseudokeronopsis as an example, we assessed the potential of SSU-V4 rDNA and four other nuclear and mitochondrial markers for species delimitation and phylogenetic reconstruction. Our studies revealed that SSU-V4 rDNA is too conservative to distinguish species, and a threshold of 97% and 99% sequence similarity detected only one and three OTUs, respectively, from seven species. On the basis of the comparative analysis of the present and previously published data, we proposed the multilocus marker including the nuclear 5.8S rDNA combining the internal transcribed spacer regions (ITS1-5.8S-ITS2) and the hypervariable D2 region of large subunit rDNA (LSU-D2) as an ideal barcode rather than the mitochondrial cytochrome c oxidase subunit 1 gene, and the ITS1-5.8S-ITS2 as a candidate metabarcoding marker for ciliates. Furthermore, the compensating base change and tree-based criteria of ITS2 and LSU-D2 were useful in complementing the DNA barcoding and metabarcoding methods by giving second structure and phylogenetic evidence.

scholarly journals PfPK6, a novel cyclin-dependent kinase/mitogen-activated protein kinase-related protein kinase from Plasmodium falciparum

2000 ◽  
Vol 347 (1) ◽  
pp. 255-263 ◽  
Author(s):  
Valerie BRACCHI-RICARD ◽  
Sailen BARIK ◽  
Cherie DELVECCHIO ◽  
Christian DOERIG ◽  
Ratna CHAKRABARTI ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Sequence Similarity ◽  
Small Subunit ◽  
Mitogen Activated Protein Kinase ◽  
Cdna Clones ◽  
Cyclin Dependent Kinase ◽  
Mitogen Activated Protein

We have isolated a novel protein kinase cDNA, PfPK6, by differential display RT-PCR (DDRT-PCR) of mRNA obtained from different asexual erythrocytic stages of Plasmodium falciparum, which shows sequence similarity to both cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) family members. The 915 bp open reading frame (ORF) is interrupted by seven introns and encodes a 305-residue polypeptide with a predicted molecular mass of 35848 Da. Several cDNA clones with some of the intron sequences were isolated, indicating alternate or defective splicing of PfPK6 transcripts because the gene seems to be a single copy located on chromosome 13. The similarity of the catalytic domain of PfPK6 to those of CDK2 and MAPK is 57.3% and 49.6%, respectively. The signature PSTAIRE (single-letter amino acid codes) CDK motif is changed to SKCILRE in PfPK6. The TXY residues that are phosphorylated in MAPKs for their activation are T173PT in PfPK6. Three size classes of PfPK6 transcripts of 6.5, 2.0 and 1.1 kb are up-regulated during the transition of P. falciparum from ring to trophozoite. Western blot analysis suggested the expression of a 35 kDa polypeptide in trophozoites and schizonts. Immunofluorescence studies indicated both nuclear and cytoplasmic localization of PfPK6 in trophozoite, schizont and segmenter stages. In vitro, recombinant PfPK6 phosphorylated itself and also exogenous substrates, histone and the small subunit of the malarial ribonucleotide reductase (R2). The kinase activity of PfPK6 is sensitive to CDK inhibitors such as olomoucine and roscovitine. PfPK6 showed a preference for Mn2+ over Mg2+ ions as a cofactor. The Lys38 → Arg mutant is severely defective in its interaction with ATP and bivalent cations and somewhat defective in catalytic rate for R2 phosphorylation.

scholarly journals The “Green” Form I Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from the Nonsulfur Purple BacteriumRhodobacter capsulatus

1999 ◽  
Vol 181 (13) ◽  
pp. 3935-3941 ◽  
Author(s):  
Kempton M. Horken ◽  
F. Robert Tabita
Keyword(s):  
Genetic Manipulation ◽  
Sequence Similarity ◽  
Large Subunit ◽  
Small Subunit ◽  
Amino Acid Sequences ◽  
Kinetic Properties ◽  
Phylogenetic Groups ◽  
Bisphosphate Carboxylase ◽  
Related Organism ◽  
Green Form

ABSTRACT Form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) of the Calvin-Benson-Bassham cycle may be divided into two broad phylogenetic groups, referred to as red-like and green-like, based on deduced large subunit amino acid sequences. Unlike the form I enzyme from the closely related organism Rhodobacter sphaeroides, the form I RubisCO from R. capsulatus is a member of the green-like group and closely resembles the enzyme from certain chemoautotrophic proteobacteria and cyanobacteria. As the enzymatic properties of this type of RubisCO have not been well studied in a system that offers facile genetic manipulation, we purified theR. capsulatus form I enzyme and determined its basic kinetic properties. The enzyme exhibited an extremely low substrate specificity factor, which is congruent with its previously determined sequence similarity to form I enzymes from chemoautotrophs and cyanobacteria. The enzymological results reported here are thus strongly supportive of the previously suggested horizontal gene transfer that most likely occurred between a green-like RubisCO-containing bacterium and a predecessor to R. capsulatus. Expression results from hybrid and chimeric enzyme plasmid constructs, made with large and small subunit genes fromR. capsulatus and R. sphaeroides, also supported the unrelatedness of these two enzymes and were consistent with the recently proposed phylogenetic placement of R. capsulatus form I RubisCO. The R. capsulatus form I enzyme was found to be subject to a time-dependent fallover in activity and possessed a high affinity for CO2, unlike the closely similar cyanobacterial RubisCO, which does not exhibit fallover and possesses an extremely low affinity for CO2. These latter results suggest definite approaches to elucidate the molecular basis for fallover and CO2 affinity.

Photoregulation of the biosynthesis of ribulose bisphosphate carboxylase

Development ◽  
1984 ◽  
Vol 83 (Supplement) ◽  
pp. 163-178
Author(s):  
R. John Ellis ◽  
Thomas F. Gallagher ◽  
Gareth I. Jenkins ◽  
C. Ruth Lennox
Keyword(s):  
Chloroplast Development ◽  
Higher Plants ◽  
Large Subunit ◽  
Nuclear Genome ◽  
Small Subunit ◽  
Ribulose Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate ◽  
Bisphosphate Carboxylase ◽  
Subunit Mrna ◽  
Parallel Increase

Chloroplast development in higher plants is light dependent, and is accompanied by the synthesis of chlorophyll and the accumulation of many chloroplast polypeptides. There is a 100-fold greater content of the photosynthetic enzyme, ribulose-1,5-bisphosphate carboxylase-oxygenase, in light-grown seedlings of Pisum sativum than in dark-grown seedlings. Following the illumination of dark-grown seedlings, there is a parallel increase in the content of both the mRNA and the polypeptide of the small subunit of the carboxylase; this subunit is a product of the nuclear genome. The increases in the mRNA and the polypeptide of the large subunit, which is a product of the chloroplast genome, show less synchronicity. Studies with isolated leaf nuclei show that the increase in small subunit mRNA is mediated primarily at the level of transcription. Three distinct effects of light on transcription of small subunit genes have been found; a rapid (∼1 h) burst, followed by a decline, when etiolated plants are first exposed to light; a slow (∼36h) development of the competence to transcribe rapidly after the initial burst; rapid (∼20 min) switches in both directions when fully greened plants are exposed to light—dark transitions.

scholarly journals Archaeal DNA Replication: Identifying the Pieces to Solve a Puzzle

Genetics ◽  
1999 ◽  
Vol 152 (4) ◽  
pp. 1249-1267 ◽  
Author(s):  
Isaac K O Cann ◽  
Yoshizumi Ishino
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Pyrococcus Furiosus ◽  
Sequence Similarity ◽  
Large Subunit ◽  
Dna Polymerases ◽  
Small Subunit ◽  
Accessory Proteins ◽  
Pol Ii

Abstract Archaeal organisms are currently recognized as very exciting and useful experimental materials. A major challenge to molecular biologists studying the biology of Archaea is their DNA replication mechanism. Undoubtedly, a full understanding of DNA replication in Archaea requires the identification of all the proteins involved. In each of four completely sequenced genomes, only one DNA polymerase (Pol BI proposed in this review from family B enzyme) was reported. This observation suggested that either a single DNA polymerase performs the task of replicating the genome and repairing the mutations or these genomes contain other DNA polymerases that cannot be identified by amino acid sequence. Recently, a heterodimeric DNA polymerase (Pol II, or Pol D as proposed in this review) was discovered in the hyperthermophilic archaeon, Pyrococcus furiosus. The genes coding for DP1 and DP2, the subunits of this DNA polymerase, are highly conserved in the Euryarchaeota. Euryarchaeotic DP1, the small subunit of Pol II (Pol D), has sequence similarity with the small subunit of eukaryotic DNA polymerase δ. DP2 protein, the large subunit of Pol II (Pol D), seems to be a catalytic subunit. Despite possessing an excellent primer extension ability in vitro, Pol II (Pol D) may yet require accessory proteins to perform all of its functions in euryarchaeotic cells. This review summarizes our present knowledge about archaeal DNA polymerases and their relationship with those accessory proteins, which were predicted from the genome sequences.

scholarly journals Primary structure of the human fgr proto-oncogene product p55c-fgr.

1988 ◽  
Vol 8 (1) ◽  
pp. 259-266 ◽  
Author(s):  
S Katamine ◽  
V Notario ◽  
C D Rao ◽  
T Miki ◽  
M S Cheah ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Sequence Similarity ◽  
Amino Acid Position ◽  
Nucleotide Sequence Analysis ◽  
Cdna Clones ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Barr Virus ◽  
Translational Product

Normal human c-fgr cDNA clones were constructed by using normal peripheral blood mononuclear cell mRNA as a template. Nucleotide sequence analysis of two such clones revealed a 1,587-base-pair-long open reading frame which predicted the primary amino acid sequence of the c-fgr translational product. Homology of this protein with the v-fgr translational product stretched from codons 128 to 516, where 32 differences among 388 codons were observed. Sequence similarity with human c-src, c-yes, and fyn translational products began at amino acid position 76 of the predicted c-fgr protein and extended nearly to its C-terminus. In contrast, the stretch of 75 amino acids at the N-terminus demonstrated a greatly reduced degree of relatedness to these same proteins. To verify the deduced amino acid sequence, antibodies were prepared against peptides representing amino- and carboxy-terminal regions of the predicted c-fgr translational product. Both antibodies specifically recognized a 55-kilodalton protein expressed in COS-1 cells transfected with a c-fgr cDNA expression plasmid. Moreover, the same protein was immunoprecipitated from an Epstein-Barr virus-infected Burkitt's lymphoma cell line which expressed c-fgr mRNA but not in its uninfected fgr mRNA-negative counterpart. These findings identified the 55-kilodalton protein as the product of the human fgr protooncogene.

scholarly journals Introduction of a leaky stop codon as molecular tool in Chlamydomonas reinhardtii

2020 ◽  
Author(s):  
Oliver D Caspari
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Photosynthetic Activity ◽  
Stop Codon ◽  
Nuclear Genome ◽  
Small Subunit ◽  
Fluorescent Reporter ◽  
Molecular Tool ◽  
Organellar Genomes ◽  
Single Construct ◽  
Bicistronic Expression

AbstractExpression of proteins in the chloroplast or mitochondria of the model green alga Chlamydomonas reinhardtii can be achieved by directly inserting transgenes into organellar genomes, or through nuclear expression and post-translational import. A number of tools have been developed in the literature for achieving high expression levels from the nuclear genome despite messy genomic integration and widespread silencing of transgenes. Here, recent advances in the field are combined and two systems of bicistronic expression, based on ribosome reinitiation or ribosomal skip induced by a viral 2A sequence, are compared side-by-side. Further, the small subunit of Rubisco (RBCS) was developed as a functional nuclear reporter for successful chloroplast import and restoration of photosynthesis: To be able to combine RBCS with a Venus fluorescent reporter without compromising photosynthetic activity, a leaky stop codon is introduced as a novel molecular tool that allows the simultaneous expression of functional and fluorescently tagged versions of the protein from a single construct.

Primary structure of the human fgr proto-oncogene product p55c-fgr

1988 ◽  
Vol 8 (1) ◽  
pp. 259-266
Author(s):  
S Katamine ◽  
V Notario ◽  
C D Rao ◽  
T Miki ◽  
M S Cheah ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Sequence Similarity ◽  
Amino Acid Position ◽  
Nucleotide Sequence Analysis ◽  
Cdna Clones ◽  
Peripheral Blood Mononuclear ◽  
Normal Peripheral Blood ◽  
Barr Virus ◽  
Translational Product

Normal human c-fgr cDNA clones were constructed by using normal peripheral blood mononuclear cell mRNA as a template. Nucleotide sequence analysis of two such clones revealed a 1,587-base-pair-long open reading frame which predicted the primary amino acid sequence of the c-fgr translational product. Homology of this protein with the v-fgr translational product stretched from codons 128 to 516, where 32 differences among 388 codons were observed. Sequence similarity with human c-src, c-yes, and fyn translational products began at amino acid position 76 of the predicted c-fgr protein and extended nearly to its C-terminus. In contrast, the stretch of 75 amino acids at the N-terminus demonstrated a greatly reduced degree of relatedness to these same proteins. To verify the deduced amino acid sequence, antibodies were prepared against peptides representing amino- and carboxy-terminal regions of the predicted c-fgr translational product. Both antibodies specifically recognized a 55-kilodalton protein expressed in COS-1 cells transfected with a c-fgr cDNA expression plasmid. Moreover, the same protein was immunoprecipitated from an Epstein-Barr virus-infected Burkitt's lymphoma cell line which expressed c-fgr mRNA but not in its uninfected fgr mRNA-negative counterpart. These findings identified the 55-kilodalton protein as the product of the human fgr protooncogene.

scholarly journals The state of oligomerization of Rubisco controls the rate of LSU translation in Chlamydomonas reinhardtii

2020 ◽  
Author(s):  
Wojciech Wietrzynski ◽  
Eleonora Traverso ◽  
Francis-André Wollman ◽  
Katia Wostrikoff
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Large Subunit ◽  
Small Subunit ◽  
Bisphosphate Carboxylase ◽  
Photosynthetic Organisms ◽  
Initiation Of Translation ◽  
Key Enzyme ◽  
Assembly Intermediate ◽  
Life On Earth

AbstractRibulose 1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) is a key enzyme for photosynthesis-driven life on Earth. While present in all photosynthetic organisms, its most prominent form is a hetero-oligomer in which a Small Subunit (SSU) stabilizes the core of the enzyme built from Large Subunits (LSU), yielding, after a chaperone-assisted multistep assembly, a LSU8SSU8 hexadecameric holoenzyme. Here we use Chlamydomonas reinhardtii, and a combination of site-directed mutants, to dissect the multistep biogenesis pathway of Rubisco in vivo. We identify assembly intermediates, in two of which LSU is associated with the RAF1 chaperone. Using genetic and biochemical approaches we further unravel a major regulation process during Rubisco biogenesis which places translation of its large subunit under the control of its ability to assemble with the small subunit, by a mechanism of Control by Epistasy of Synthesis (CES). Altogether this leads us to propose a model where the last assembly intermediate, an octameric LSU8-RAF1 complex which delivers LSU to SSU to form the Rubisco enzyme, converts to a key regulator form able to exert a negative feed-back on the initiation of translation of LSU, when SSU is not available.

scholarly journals Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6

2006 ◽  
Vol 72 (3) ◽  
pp. 1825-1832 ◽  
Author(s):  
Mikio Sasoh ◽  
Eiji Masai ◽  
Satoko Ishibashi ◽  
Hirofumi Hara ◽  
Naofumi Kamimura ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Large Subunit ◽  
Transcriptional Regulator ◽  
Gene Clusters ◽  
Small Subunit ◽  
Amino Acid Sequence Similarity ◽  
Comamonas Testosteroni ◽  
Cell Extracts ◽  
Two Component

ABSTRACT We isolated Comamonas sp. strain E6, which utilizes terephthalate (TPA) as the sole carbon and energy source via the protocatechuate (PCA) 4,5-cleavage pathway. Two almost identical TPA degradation gene clusters, tphR I C I A2 I A3 I B I A1 I and tphR II C II A2 II A3 II B II A1 II, were isolated from this strain. Based on amino acid sequence similarity, the genes tphR, tphC, tphA2, tphA3, tphB, and tphA1 were predicted to code, respectively, for an IclR-type transcriptional regulator, a periplasmic TPA binding receptor, the large subunit of the oxygenase component of TPA 1,2-dioxygenase (TPADO), the small subunit of the oxygenase component of TPADO, a 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate (DCD) dehydrogenase, and a reductase component of TPADO. The growth of E6 on TPA was not affected by disruption of either tphA2 I or tphA2 II singly; however, the tphA2 I tphA2 II double mutant no longer grew on TPA, suggesting that both TPADO genes are involved in TPA degradation. Introduction of a plasmid carrying tphR II C II A2 II A3 II B II A1 II conferred the TPA utilization phenotype on Comamonas testosteroni IAM 1152, which is able to grow on PCA but not on TPA. Disruption of either tphR II or tphC II on this plasmid resulted in the loss of the growth of IAM 1152 on TPA, suggesting that these genes are essential to convert TPA to PCA in E6. The genes tphA1 II, tphA2 II, tphA3 II, and tphB II were expressed in Escherichia coli, and the resultant cell extracts containing TphA1II, TphA2II, and TphA3II converted TPA in the presence of NADPH into a product which was transformed to PCA by TphBII. On the basis of these results, TPADO was strongly suggested to be a two-component dioxygenase which consists of the terminal oxygenase component (TphA2 and TphA3) and the reductase (TphA1), and tphB codes for the DCD dehydrogenase.

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