Continuous chlorophyll degradation accompanied by chlorophyllide and phytol reutilization for chlorophyll synthesis in Synechocystis sp. PCC 6803

Chlorophyll synthesis and degradation were analyzed in the cyanobacterium Synechocystis sp. PCC 6803 by incubating cells in the presence of ¹³C-labeled glucose or ¹⁵N-containing salts. Upon mass spectral analysis of chlorophyll isolated from cells grown in the presence of ¹³C-glucose for different time periods, four chlorophyll pools were detected that differed markedly in the amount of ¹³C incorporated into the porphyrin (Por) and phytol (Phy) moieties of the molecule. These four pools represent (i) unlabeled chlorophyll (¹²Por¹²Phy), (ii) ¹³C-labeled chlorophyll (¹³Por¹³Phy), and (iii, iv) chlorophyll, in which either the porphyrin or the phytol moiety was ¹³C-labeled, whereas the other constituent of the molecule remained unlabeled (¹³Por¹²Phy and ¹²Por¹³Phy). The kinetics of ¹²Por¹²Phy disappearance, presumably due to chlorophyll de-esterification, and of ¹³Por¹²Phy, ¹²Por¹³Phy, and ¹³Por¹³Phy accumulation due to chlorophyll synthesis provided evidence for continuous chlorophyll turnover in Synechocystis cells. The loss of ¹²Por¹²Phy was three-fold faster in a photosystem I-less strain than in a photosystem II-less strain and was accelerated in wild-type cells upon exposure to strong light. These data suggest that most chlorophyll appears to be de-esterified in Synechocystis upon dissociation and repair of damaged photosystem II. A substantial part of chlorophyllide and phytol released upon the de-esterification of chlorophyll can be recycled for the biosynthesis of new chlorophyll molecules contributing to the formation of ¹³Por¹²Phy and ¹²Por¹³Phy chlorophyll pools. The phytol kinase, Slr1652, plays a significant but not absolutely critical role in this recycling process.     

Acyclic 2′, 3′-Dideoxy-2′,3′-didehydronucleoside 5′-Triphosphates as Termination Substrates of Broad Set of DNA Polymerases

Abstract

O4′-Nor-2′,3′-dideoxy-2′,3′-didehydronucleoside 5′-triphosphates (acyclo-d₄NTP) have been shown to have the properties of effective termination substrates for DNA biosynthesis, catalyzed by several different DNA polymerases.     

Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations in CDC28

Current eukaryotic replication models postulate that leading and lagging DNA strands are replicated predominantly by dedicated DNA polymerases. The catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable, while the inactive C-terminal part is required for viability. Despite extensive studies of yeast Saccharomyces cerevisiae strains lacking the active N-terminal half, it is still unclear how these strains survive and recover. We designed a robust method for constructing mutants with only the C-terminal part of Pol2. Strains without the active polymerase part show severe growth defects, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly accumulate fast-growing clones. Analysis of genomic DNA sequences of these clones revealed that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs by a positive selection of mutants with improved growth. Elevated mutation rates help generate sufficient numbers of these variants. Single nucleotide changes in the cell cycle-dependent kinase gene, CDC28, improve the growth of strains lacking the N-terminal part of Pol2, and rescue their sensitivity to replication inhibitors and, in parallel, lower mutation rates. Our study predicts that changes in mammalian homologs of cyclin-dependent kinases may contribute to cellular responses to the leading strand polymerase defects.     

Chaos in a seasonally perturbed SIR model: avian influenza in a seabird colony as a paradigm

Seasonality is a complex force in nature that affects multiple processes in wild animal populations. In particular, seasonal variations in demographic processes may considerably affect the persistence of a pathogen in these populations. Furthermore, it has been long observed in computer simulations that under seasonal perturbations, a host–pathogen system can exhibit complex dynamics, including the transition to chaos, as the magnitude of the seasonal perturbation increases. In this paper, we develop a seasonally perturbed Susceptible-Infected-Recovered model of avian influenza in a seabird colony. Numerical simulations of the model give rise to chaotic recurrent epidemics for parameters that reflect the ecology of avian influenza in a seabird population, thereby providing a case study for chaos in a host– pathogen system. We give a computer-assisted exposition of the existence of chaos in the model using methods that are based on the concept of topological hyperbolicity. Our approach elucidates the geometry of the chaos in the phase space of the model, thereby offering a mechanism for the persistence of the infection. Finally, the methods described in this paper may be immediately extended to other infections and hosts, including humans.     

G-specific RNA-cleaving Conjugates of Short Peptides and Oligodeoxyribonucleotides

Abstract

Artificial ribonucleases, conjugates of short oligodeoxyribonucleotides and peptides built of arginine, leucine, proline, and serine, were synthesized and assessed in terms of ribonuclease activity and specificity of RNA cleavage. A specific group of the conjugates was identified that display T1-ribonuclease-like activity and cleave RNA predominantly at G-X sequences. Circular dichroism study of the structures of the most active conjugates, free peptide (LR)4G, and oligonucleotides revealed that conjugation of oligonucleotide to the peptide results in a specific peptide folding that possibly provides ribonuclease activity to the conjugate.     

The Terpolymer Produced by Azotobacter Chroococcum 7B: Effect of Surface Properties on Cell Attachment

The copolymerization of poly(3-hydroxybutyrate) (PHB) is a promising trend in bioengineering to improve biomedical properties, e.g. biocompatibility, of this biodegradable polymer. We used strain Azotobacter chroococcum 7B, an effective producer of PHB, for biosynthesis of not only homopolymer and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also novel terpolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-poly(ethylene glycol) (PHB-HV-PEG), using sucrose as the primary carbon source and valeric acid and poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-HV-PEG was confirmed by ¹H nuclear-magnetic resonance analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) of produced biopolymer, the protein adsorption to the terpolymer, and cell growth on biopolymer films were studied. Despite of low EG-monomers content in bacterial-origin PHB-HV-PEG polymer, the terpolymer demonstrated significant improvement in biocompatibility in vitro in contrast to PHB and PHB-HV polymers, which may be coupled with increased protein adsorption, hydrophilicity and surface roughness of PEG-containing copolymer.     

An integrated chromosome map of microsatellite markers and inversion breakpoints for an Asian malaria mosquito, Anopheles stephensi

Anopheles stephensi is one of the major vectors of malaria in the Middle East and Indo-Pakistan subcontinent. Understanding the population genetic structure of malaria mosquitoes is important for developing adequate and successful vector control strategies. Commonly used markers for inferring anopheline taxonomic and population status include microsatellites and chromosomal inversions. Knowledge about chromosomal locations of microsatellite markers with respect to polymorphic inversions could be useful for better understanding a genetic structure of natural populations. However, fragments with microsatellites used in population genetic studies are usually too short for successful labeling and hybridization with chromosomes. We designed new primers for amplification of microsatellite loci identified in the A. stephensi genome sequenced with next-generation technologies. Twelve microsatellites were mapped to polytene chromosomes from ovarian nurse cells of A. stephensi using fluorescent in situ hybridization. All microsatellites hybridized to unique locations on autosomes, and 7 of them localized to the largest arm 2R. Ten microsatellites were mapped inside the previously described polymorphic chromosomal inversions, including 4 loci located inside the widespread inversion 2Rb. We analyzed microsatellite-based population genetic data available for A. stephensi in light of our mapping results. This study demonstrates that the chromosomal position of microsatellites may affect estimates of population genetic parameters and highlights the importance of developing physical maps for nonmodel organisms.     

Continuous chlorophyll degradation accompanied by chlorophyllide and phytol reutilization for chlorophyll synthesis in Synechocystis sp. PCC 6803

Chlorophyll synthesis and degradation were analyzed in the cyanobacterium Synechocystis sp. PCC 6803 by incubating cells in the presence of 13C-labeled glucose or 15N-containing salts. Upon mass spectral analysis of chlorophyll isolated from cells grown in the presence of 13C-glucose for different time periods, four chlorophyll pools were detected that differed markedly in the amount of 13C incorporated into the porphyrin (Por) and phytol (Phy) moieties of the molecule. These four pools represent (i) unlabeled chlorophyll (12Por12Phy), (ii) 13C-labeled chlorophyll (13Por13Phy), and (iii, iv) chlorophyll, in which either the porphyrin or the phytol moiety was 13C-labeled, whereas the other constituent of the molecule remained unlabeled (13Por12Phy and 12Por13Phy). The kinetics of 12Por12Phy disappearance, presumably due to chlorophyll de-esterification, and of 13Por12Phy, 12Por13Phy, and 13Por13Phy accumulation due to chlorophyll synthesis provided evidence for continuous chlorophyll turnover in Synechocystis cells. The loss of 12Por12Phy was three-fold faster in a photosystem I-less strain than in a photosystem II-less strain and was accelerated in wild-type cells upon exposure to strong light. These data suggest that most chlorophyll appears to be de-esterified in Synechocystis upon dissociation and repair of damaged photosystem II. A substantial part of chlorophyllide and phytol released upon the de-esterification of chlorophyll can be recycled for the biosynthesis of new chlorophyll molecules contributing to the formation of 13Por12Phy and 12Por13Phy chlorophyll pools. The phytol kinase, Slr1652, plays a significant but not absolutely critical role in this recycling process.     

The evolution of gene regulatory networks controlling Arabidopsis thaliana L. trichome development

Background

The variation in structure and function of gene regulatory networks (GRNs) participating in organisms development is a key for understanding species-specific evolutionary strategies. Even the tiniest modification of developmental GRN might result in a substantial change of a complex morphogenetic pattern. Great variety of trichomes and their accessibility makes them a useful model for studying the molecular processes of cell fate determination, cell cycle control and cellular morphogenesis. Nowadays, a large number of genes regulating the morphogenesis of A. thaliana trichomes are described. Here we aimed at a study the evolution of the GRN defining the trichome formation, and evaluation its importance in other developmental processes.

Results

In study of the evolution of trichomes formation GRN we combined classical phylogenetic analysis with information on the GRN topology and composition in major plants taxa. This approach allowed us to estimate both times of evolutionary emergence of the GRN components which are mainly proteins, and the relative rate of their molecular evolution. Various simplifications of protein structure (based on the position of amino acid residues in protein globula, secondary structure type, and structural disorder) allowed us to demonstrate the evolutionary associations between changes in protein globules and speciations/duplications events. We discussed their potential involvement in protein-protein interactions and GRN function.

Conclusions

We hypothesize that the divergence and/or the specialization of the trichome-forming GRN is linked to the emergence of plant taxa. Information about the structural targets of the protein evolution in the GRN may predict switching points in gene networks functioning in course of evolution. We also propose a list of candidate genes responsible for the development of trichomes in a wide range of plant species.

     

Study of electron-dense thylakoids in chloroplasts of Stevia rebaudiana Bertoni in relation to the biosynthesis of diterpenoids

Using transmission electronic microscopy and mass spectrometry electron-dense thylakoids of chloroplasts of Stevia rebaudiana leaves during active vegetable growth of this plant were studied in relation to the biosynthesis of diterpenoid glycosides (DGs). It was found that these compounds are absent in these thylakoids, but they contain a water-insoluble weakly polar ent-kauren, a known biosynthetic precursor of DGs as well as gibberellins. This finding provides a base for the suggestion that similar, electron-dense, thylakoids were observed earlier by other authors in other plant species. These data allowed us to conclude that an intensive biosynthesis of ent-kauren is likely related to adaptation of the short-day plants including Stevia rebaudiana to vegetable growth under the long day conditions.