Intercistronic expression elements (IEE) from the chloroplast of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> can be used for the expression of foreign genes in synthetic operons

Key message

Two intercistronic regions were identified as functional intercistronic expression elements (IEE) for the simultaneous expression of aphA-6 and gfp in a synthetic operon in the chloroplast of C. reinhardtii.

Abstract

Chlamydomonas reinhardtii, a biflagellate photosynthetic microalga, has been widely used in basic and applied science. Already three decades ago, Chlamydomonas had its chloroplast genome transformed and to this day constitutes the only alga routinely used in transplastomic technology. Despite the fact that over a 100 foreign genes have been expressed from the chloroplast genome, little has been done to address the challenge of expressing multiple genes in the form of operons, a development that is needed and crucial to push forward metabolic engineering and synthetic biology in this organism. Here, we studied five intercistronic regions and investigated if they can be used as intercistronic expression elements (IEE) in synthetic operons to drive the expression of foreign genes in the chloroplast of C. reinhardtii. The intercistronic regions were those from the psbB-psbT, psbN-psbH, psaC-petL, petL-trnN and tscA-chlN chloroplast operons, and the foreign genes were the aminoglycoside 3′-phosphotransferase (aphA-6), which confers resistance to kanamycin, and the green fluorescent protein gene (gfp). While all the intercistronic regions yielded lines that were resistant to kanamycin, only two (obtained with intercistronic regions from psbN-psbH and tscA-chlN) were identified as functional IEEs, yielding lines in which the second cistron (gfp) was translated and generated GFP. The IEEs we have identified could be useful for the stacking of genes for metabolic engineering or synthetic biology circuits in the chloroplast of C. reinhardtii.

     

Three molecular markers suggest different relationships among three <Emphasis Type="Italic">Drepanocladus</Emphasis> species (Bryophyta: Amblystegiaceae)

Relationships among numerous specimens of Drepanocladus angustifolius (35 specimens), Drepanocladus lycopodioides (71 specimens), and Drepanocladus turgescens (102 specimens) are analysed based on the nuclear internal transcribed spacers 1 and 2 (ITS) and a portion of glyceraldehyde 3-phosphate dehydrogenase (gpd), and the plastid rpl16 G2 intron. Molecular data suggest that neither species is monophyletic as well as significant incongruence among molecular markers. No statistical support for recombination (ITS, gpd) was found. For some D. lycopodioides and D. turgescens specimens, the molecular information even suggests that they belong to the wrong one of these two species. All such specimens were collected in south Swedish areas where the two species are frequently found growing together and where sporophytes are often common. The occurrence of all such specimens only in these geographical areas is statistically unlikely and suggests that hybridisation occasionally occurs here. While molecular information suggests that the species are not monophyletic, geographical signals could be found for both D. angustifolius and D. turgescens.     

Effect of codon optimization on the enhancement of the β-carotene contents in rice endosperm

A β-carotene is the most well-known dietary source as provitamin A carotenoids. Among β-carotene-producing Golden Rice varieties, PAC (Psy:2A:CrtI) rice has been previously developed using a bicistronic recombinant gene that linked the Capsicum Psy and Pantoea CrtI genes by a viral 2A sequence. To enhance β-carotene content by improving this PAC gene, its codon was optimized for rice plants (Oryza sativa L.) by minimizing the codon bias between the transgene donor and the host rice and was then artificially synthesized as stPAC (stPsy:2A:stCrtI) gene. The GC content (58.7 from 50.9%) and codon adaptation index (0.85 from 0.77) of the stPAC gene were increased relative to the original PAC gene with 76% DNA identity. Among 67 T₁ seeds of stPAC transformants showing positive correlations between transgene copy numbers (up to three) and carotenoid contents, three stPAC lines with a single intact copy were chosen to minimize unintended insertional effects and compared to the representative line of the PAC transgene with respect to their codon optimization effects. Translation levels were stably increased in all three stPAC lines (3.0-, 2.5-, 2.9-fold). Moreover, a greater intensity of the yellow color of stPAC seeds was correlated with enhanced levels of β-carotene (4-fold, 2.37 μg/g) as well as total carotenoid (2.9-fold, 3.50 μg/g) relative to PAC seeds, suggesting a β-branch preference for the stPAC gene. As a result, the codon optimization of the transgene might be an effective tool in genetic engineering for crop improvement as proven at the enhanced levels of translation and carotenoid production.     

Gene Expression Prediction and Hierarchical Clustering Analysis of Plant <Emphasis Type="Italic">CCD</Emphasis> genes

Plant carotenoid cleavage dioxygenase (CCD) catalyses the formation of industrially important apocarotenoids. Here, we applied codon-based classification for 72 CCD genes from 35 plant species using hierarchical clustering analysis. The codon adaptation index (CAI) and relative codon bias (RCB) were utilized to estimate the level of gene expression. The codon-based cluster tree result shows neatly clustered subclass of CCD genes except BoCCD1 gene of Bixa orellana. Correlation analysis of CAI values with RCB indicates an overall low-level expression of CCD across different species. Similarly, the closeness in the codon cluster with same CAI values was not reflected in 3-D structural report of selected CCD genes. These finding not only enhances our insights into the classification of CCD gene across the species but also identifies the critical factors responsible for this variation, which could aid in prediction of gene expression and function for newly reported CCD genes.     

Analysis of synonymous codon usage bias in <Emphasis Type="Italic">helicase</Emphasis> gene from <Emphasis Type="Italic">Autographa californica</Emphasis> multiple nucleopolyhedrovirus

The helicase gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is not only involved in viral DNA replication, but also plays a role in viral host range. To identify the codon usage bias of helicase of AcMNPV, the codon usage bias of helicase was especially studies in AcMNPV and 41 reference strains of baculoviruses by calculating the codon adaptation index (CAI), effective number of codon (ENc), relative synonymous codon usage (RSCU), and other indices. The helicase of baculovirus is less biased (mean ENc?=?50.539?>?40; mean CAI?=?0.246). AcMNPV helicase has a strong bias toward the synonymous codons with G and C at the third codon position (GC3s?=?53.6%). The plot of GC3s against ENc values revealed that GC compositional constraints are the main factor that determines the codon usage bias of major of helicase. Several indicators supported that the codon usage pattern of helicase is mainly subject to mutation pressure. Analysis of variation in codon usage and amino acid composition indicated AcMNPV helicase shows the significant preference for one or more postulated codons for each amino acid. A cluster analysis based on RSCU values suggested that AcMNPV is evolutionarily closer to members of group I alphabaculovirus. Comparison of the codon usage pattern among E. coli, yeast, mouse, human and AcMNPV showed that yeast is a suitable expression system for AcMNPV helicase. AcMNPV helicase shows weak codon usage bias. This study may help in elucidating the functional mechanism of AcMNPV helicase and the evolution of baculovirus helicases.     

Differentiation of the endemic Greek genus <Emphasis Type="Italic">Hymenonema</Emphasis> and its relatives of subtribe Scolyminae (Compositae,Cichorieae) based on a multilocus species tree reconstruction

Hymenonema (Compositae, tribe Cichorieae) together with the genera Catananche, Gundelia, and Scolymus forms the subtribe Scolyminae. It is endemic to Greece and consists of two species, Hymenonema laconicum and Hymenonema graecum, which occur in the south Peloponnisos and central Aegean area, respectively. The present contribution aims at a phylogenetic reconstruction of evolutionary relationships among the 12 species of the subtribe, focusing on the temporal and spatial framework for its evolution. The phylogenetic relationships among the members of Scolyminae were inferred from molecular data based on the multi-copy region of the nrDNA internal transcribed spacers ITS1 and ITS2, two intergenic spacers of the cpDNA (trnL-trnF, rpl32-trnL), and one single-copy nuclear region (D10). The gene trees were reconstructed using Bayesian phylogenetic methods. All gene trees support the monophyly of Hymenonema and the sister-group relationship with the genus Scolymus. The further sister-group relationship of this group (Hymenonema–Scolymus) with Catananche is also supported by nrDNA and cpDNA analyses. Finally, a species tree (inferred in a Bayesian coalescent framework) was reconstructed and dates the divergence time between the two Hymenonema species to the Pleistocene (around 1.3 Ma ago). Maximum likelihood-based biogeographical reconstructions suggest a Miocene (pre-Messinian) differentiation of the subtribe on the northern Tethyan platform, followed by Miocene/Pliocene dispersal events to the western Mediterranean and North-African platforms and final, small-scale vicariance events within the genera in the Pleistocene.     

The role of gene fusions in the evolution of metabolic pathways: the histidine biosynthesis case

Background

Histidine biosynthesis is one of the best characterized anabolic pathways. There is a large body of genetic and biochemical information available, including operon structure, gene expression, and increasingly larger sequence databases. For over forty years this pathway has been the subject of extensive studies, mainly in Escherichia coli and Salmonella enterica, in both of which details of histidine biosynthesis appear to be identical. In these two enterobacteria the pathway is unbranched, includes a number of unusual reactions, and consists of nine intermediates; his genes are arranged in a compact operon (hisGDC [NB]HAF [IE]), with three of them (hisNB, hisD and hisIE) coding for bifunctional enzymes. We performed a detailed analysis of his gene fusions in available genomes to understand the role of gene fusions in shaping this pathway.

Results

The analysis of HisA structures revealed that several gene elongation events are at the root of this protein family: internal duplication have been identified by structural superposition of the modules composing the TIM-barrel protein.Several his gene fusions happened in distinct taxonomic lineages; hisNB originated within γ-proteobacteria and after its appearance it was transferred to Campylobacter species (ε-proteobacteria) and to some Bacteria belonging to the CFB group. The transfer involved the entire his operon. The hisIE gene fusion was found in several taxonomic lineages and our results suggest that it probably happened several times in distinct lineages.Gene fusions involving hisIE and hisD genes (HIS4) and hisH and hisF genes (HIS7) took place in the Eukarya domain; the latter has been transferred to some δ-proteobacteria.

Conclusion

Gene duplication is the most widely known mechanism responsible for the origin and evolution of metabolic pathways; however, several other mechanisms might concur in the process of pathway assembly and gene fusion appeared to be one of the most important and common.

     

Fingerprinting by amplified fragment length polymorphism (AFLP) and barcoding by three plastidic markers in the genus <Emphasis Type="Italic">Wolffiella</Emphasis> Hegelm

Amplified fragment length polymorphism (AFLP) fingerprinting and three different plastidic DNA regions (rpl16, rps16, atpF-atpH) were used to investigate species identity in the genus Wolffiella. For this purpose, clones (67 in total) belonging to all ten species were selected. Almost all the species were represented by more than one clone. The fingerprinting technique, AFLP, clearly distinguished the species, W. caudata, W. gladiata, W. neotropica, W. rotunda, and W. welwitschii. Apart from confirming the molecular identity of these five species, the plastidic markers could delineate two additional species, W. hyalina and W. denticulata, although the conclusion concerning the latter is restricted by the availability of only one clone. The efficiency of the plastid-derived markers in identifying the number of species-specific clusters followed the sequence rps16 > rpl16 > atpF-atpH. The species W. lingulata, W. oblonga, and W. repanda could not be identified by any of the molecular methods presented here, but could be strictly defined on a morphological basis. In several clones, high amounts of genetic admixtures between different species were detected. Further, simulation studies demonstrated that these clones are genetic hybrids. This might be one of the obstacles in molecular identification of species in the genus Wolffiella.     

<Emphasis Type="Italic">Streptomyces xiangluensis</Emphasis> sp. nov., a novel actinomycete isolated from soil

A novel actinomycete, designated strain NEAU-LA29^T, was isolated from soil collected from Xianglu Mountain and subjected to a polyphasic taxonomic study. Based on a polyphasic taxonomic approach comprising chemotaxonomic, phylogenetic, morphological and physiological characterisation, the isolate has been affiliated to the genus Streptomyces. 16S rRNA gene sequence analysis showed that the isolate is closely related to Streptomyces vastus JCM4524^T (98.8% identity) and Streptomyces cinereus DSM43033^T (97.9%). However, multilocus sequence analysis based on five other house-keeping genes (atpD, gyrB, rpoB, recA and trpB) and low DNA–DNA relatedness values enabled the strain to be differentiated from these closely related species of the genus Streptomyces. Thus, strain NEAU-LA29^T is concluded to represent a novel species of the genus Streptomyces, for which the name Streptomyces xiangluensis sp. nov. is proposed. The type strain is NEAU-LA29^T (=?CGMCC 4.7466^T?=?DSM 105786^T).     

Assembly of a complete genome sequence for <Emphasis Type="Italic">Gemmata obscuriglobus</Emphasis> reveals a novel prokaryotic rRNA operon gene architecture

Gemmata obscuriglobus is a Gram-negative bacterium with several intriguing biological features. Here, we present a complete, de novo whole genome assembly for G. obscuriglobus which consists of a single, circular 9 Mb chromosome, with no plasmids detected. The genome was annotated using the NCBI Prokaryotic Genome Annotation pipeline to generate common gene annotations. Analysis of the rRNA genes revealed three interesting features for a bacterium. First, linked G. obscuriglobus rrn operons have a unique gene order, 23S–5S–16S, compared to typical prokaryotic rrn operons (16S–23S–5S). Second, G. obscuriglobus rrn operons can either be linked or unlinked (a 16S gene is in a separate genomic location from a 23S and 5S gene pair). Third, all of the 23S genes (5 in total) have unique polymorphisms. Genome analysis of a different Gemmata species (SH-PL17), revealed a similar 23S–5S–16S gene order in all of its linked rrn operons and the presence of an unlinked operon. Together, our findings show that unique and rare features in Gemmata rrn operons among prokaryotes provide a means to better define the evolutionary relatedness of Gemmata species and the divergence time for different Gemmata species. Additionally, these rrn operon differences provide important insights into the rrn operon architecture of common ancestors of the planctomycetes.     

Phage integrases for the construction and manipulation of transgenic mammals

Phage integrases catalyze site-specific, unidirectional recombination between two short att recognition sites. Recombination results in integration when the att sites are present on two different DNA molecules and deletion or inversion when the att sites are on the same molecule. Here we demonstrate the ability of the φC31 integrase to integrate DNA into endogenous sequences in the mouse genome following microinjection of donor plasmid and integrase mRNA into mouse single-cell embryos. Transgenic early embryos and a mid-gestation mouse are reported. We also demonstrate the ability of the φC31, R4, and TP901-1 phage integrases to recombine two introduced att sites on the same chromosome in human cells, resulting in deletion of the intervening material. We compare the frequencies of mammalian chromosomal deletion catalyzed by these three integrases in different chromosomal locations. The results reviewed here introduce these bacteriophage integrases as tools for site-specific modification of the genome for the creation and manipulation of transgenic mammals.     

The mitochondrial DNA of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>: complete sequence,gene content and genome organization

We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564?bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4?kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii.     

Comparative expression in <Emphasis Type="Italic">Escherichia coli</Emphasis> of the native and hybrid genes for acyl-lipid Δ<Superscript>9</Superscript> desaturase

Expression of the desC gene coding for acyl-lipid Δ⁹ desaturase of thermophilic cyanobacterium Synechocystis sp. PCC6803 was studied in Escherichia coli cells. In a hybrid gene constructed (desC-licBM3), a sequence of the native acyl-lipid Δ⁹ desaturase was fused in frame with the reporter gene coding for thermostable lichenase. Lichenase contained in the hybrid protein simplified selection and analysis of the expression of membrane desaturase in the heterologous host. Comparisons of the expression for the native and hybrid genes in bacterial cells showed that lichenase remained active and thermostable in the hybrid protein, while desaturase retains the capability of introducing a double bound in the corresponding position of fatty acid residues.     

Salicylate degradation by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strains not involving the “Classical” <Emphasis Type="Italic">nah2</Emphasis> operon

Genetic systems for salicylate catabolism were analyzed in 12 strains of Pseudomonas putida, isolated from polluted soil samples collected in the Murmansk and Tula oblasts. All of the studied P. putida strains utilize salicylate in the ortho-pathway of catechol cleavage without employing the enzymes of the “classical” nah2 operon. The data demonstrates that salicylate degradation in the studied strains is performed with the involvement of the salicylate hydroxylase gene analogous to the nahU gene of strain P. putida ND6. New variants of salicylate hydroxylase genes nahG1 and nahU were found.     

Arabinose C Protein: Regulation of the Arabinose Operon <Emphasis Type="Italic">in vitro</Emphasis>

The detection of the gene ara C protein using a DNA-dependent in vitro protein synthesizing system represents the first isolation of an operon specific positive regulator with an in vivo role that has been genetically defined.     

Low molecular weight glutenin subunit gene composition at <Emphasis Type="Italic">Glu</Emphasis>-<Emphasis Type="Italic">D3</Emphasis> loci of <Emphasis Type="Italic">Aegilops tauschii</Emphasis> and common wheat and a further view of wheat evolution

Key message

A comprehensive comparison of LMW-GS genes between Ae. tauschii and its progeny common wheat.

Abstract

Low molecular weight glutenin subunits (LMW-GSs) are determinant of wheat flour processing quality. However, the LMW-GS gene composition in Aegilops tauschii, the wheat D genome progenitor, has not been comprehensively elucidated and the impact of allohexaploidization on the Glu-D3 locus remains elusive. In this work, using the LMW-GS gene molecular marker system and the full-length gene-cloning method, LMW-GS genes at the Glu-D3 loci of 218 Ae. tauschii and 173 common wheat (Triticum aestivum L.) were characterized. Each Ae. tauschii contained 11 LMW-GS genes, and the whole collection was divided into 25 haplotypes (AeH01–AeH25). The Glu-D3 locus in common wheat lacked the LMW-GS genes D3-417, D3-507 and D3-552, but shared eight genes of identical open reading frame (ORF) sequences when compared to that of Ae. tauschii. Therefore, the allohexaploidization induces deletions, but exerts no influence on LMW-GS gene coding sequences at the Glu-D3 locus. 92.17% Ae. tauschii had 7-9 LMW-GSs, more than the six subunits in common wheat. The haplotypes AeH16, AeH20 and AeH23 of Ae. tauschii ssp. strangulate distributed in southeastern Caspian Iran were the main putative D genome donor of common wheat. These results facilitate the utilization of the Ae. tauschii glutenin gene resources and the understanding of wheat evolution.

     

Association of genes of different functional classes with type 1 diabetes

The genetic structure of susceptibility to type 1 diabetes (T1D) in the population of Tomsk was studied. We had a group of T1D patients (N = 285) and a population sample (N = 300) and we studied 58 SNPs localized in the 47 genes which products are involved in various metabolic pathways and processes as fibrogenesis, endothelial dysfunction, and inflammation. Genotyping was performed by mass spectrometry using the Sequenom MassARRAY system (United States). We compared the group of T1D patients and the population sample and found an association with the predisposition to disease for seven markers: rs3765124 of the ADAMDEC1 gene, genotype AA (p = 0.004), allele A (p = 0.033); rs1007856 of the ITGB5 gene, genotype TT (p = 0.015), allele T (p = 0.036); rs20579 of the LIG1 gene, genotype CC (p = 0.004), allele C (p = 0.002); rs12980602 of the IFNL2 gene, allele C (p = 0.029); rs4986819 of the PARP4 gene, allele C (p = 0.044); rs1143674 of the ITGA4 gene genotype GG (p = 0.002); rs679620 of the MMP3 gene, genotype AA (p = 0.008). Thus, the products of genes associated with T1D belong to different molecular classes: metalloproteases (ADAMDEC1, MMP3), cytokines (IL28A), cell surface receptors (ITGA4), adhesion molecules (ITGB5), DNA ligases (LIG1), and ribosyltransferase enzymes (PARP4). The ADAMDEC1, ITGA4, and ITGB5 genes belong to two biological processes: cell communication and signal transduction. The LIG1 and PARP4 genes regulate the metabolism of nucleic acids, MMP3 is involved in the regulation of protein metabolism, and the IFNL2 is involved in the immune response.     

Two new complete mitochondrial genomes of <Emphasis Type="Italic">Dorcus</Emphasis> stag beetles (Coleoptera,Lucanidae)

The systematics of Dorcus MacLeay has been a long-standing debate. Mitochondrial genomes were widely used to deeply understand the phylogeny of problematic taxa in virtue of their genetic importance and comprehensiveness. To provide more useful genetic data for resolving the systematic disputation of Dorcus stag beetles. The complete mitochondrial genomes of Dorcus hopei and Dorcus seguyi were obtained using the next generation sequencing. Characteristics of the two genomes are explicated through comparing their genome organization and base composition, protein-coding genes and codon usage, intergenic spacers and non-coding region, transfer and ribosomal RNA genes and control region. Phylogenetic relationships were reconstructed using Maximum likelihood and Bayesian inference analyses based on the concatenated nucleotide sequences of 13 PCGs from 9 stag beetles and 3 scarab beetles. The complete mitogenomes of D. hopei and D. seguyi was 16,026 bp/17,955 bp long, respectively. A tandem repeat with the length of 940 bp was presented in the A+T-rich region in D. hopei. An unexpected non-coding region of 332 bp was located between nad2 and trnW in D. seguyi. The phylogenetic analyses robustly supported that D. hopei formed a branch with the generic type of D. parallelipipedus. Whereas D. seguyi was not covered in the branch of (D. hopei?+?D. parallelipipedus), but was sister to them. The results indicated that D. hopei should be a good member of Dorcus MacLeay. The taxonomic status of D. seguyi remained to be studied furtherly.