Challenges of the Human Proteome Project: 10-Year Experience of the Russian Consortium.

This manuscript collects all the efforts of the Russian Consortium, bottlenecks revealed in the course of the C-HPP realization, and ways of their overcoming. One of the main bottlenecks in the C-HPP is the insufficient sensitivity of proteomic technologies, hampering the detection of low- and ultralow-copy number proteins forming the "dark part" of the human proteome. In the frame of MP-Challenge, to increase proteome coverage we suggest an experimental workflow based on a combination of shotgun technology and selected reaction monitoring with two-dimensional alkaline fractionation. Further, to detect proteins that cannot be identified by such technologies, nanotechnologies such as combined atomic force microscopy with molecular fishing and/or nanowire detection may be useful. These technologies provide a powerful tool for single molecule analysis, by analogy with nanopore sequencing during genome analysis. To systematically analyze the functional features of some proteins (CP50 Challenge), we created a mathematical model that predicts the number of proteins differing in amino acid sequence: proteoforms. According to our data, we should expect about 100 000 different proteoforms in the liver tissue and a little more in the HepG2 cell line. The variety of proteins forming the whole human proteome significantly exceeds these results due to post-translational modifications (PTMs). As PTMs determine the functional specificity of the protein, we propose using a combination of gene-centric transcriptome-proteomic analysis with preliminary fractionation by two-dimensional electrophoresis to identify chemically modified proteoforms. Despite the complexity of the proposed solutions, such integrative approaches could be fruitful for MP50 and CP50 Challenges in the framework of the C-HPP.

New insights from Opisthorchis felineus genome: update on genomics of the epidemiologically important liver flukes.

BACKGROUND: The three epidemiologically important Opisthorchiidae liver flukes Opisthorchis felineus, O. viverrini, and Clonorchis sinensis, are believed to harbour similar potencies to provoke hepatobiliary diseases in their definitive hosts, although their populations have substantially different ecogeographical aspects including habitat, preferred hosts, population structure. Lack of O. felineus genomic data is an obstacle to the development of comparative molecular biological approaches necessary to obtain new knowledge about the biology of Opisthorchiidae trematodes, to identify essential pathways linked to parasite-host interaction, to predict genes that contribute to liver fluke pathogenesis and for the effective prevention and control of the disease. RESULTS: Here we present the first draft genome assembly of O. felineus and its gene repertoire accompanied by a comparative analysis with that of O. viverrini and Clonorchis sinensis. We observed both noticeably high heterozygosity of the sequenced individual and substantial genetic diversity in a pooled sample. This indicates that potency of O. felineus population for rapid adaptive response to control and preventive measures of opisthorchiasis is higher than in O. viverrini and C. sinensis. We also have found that all three species are characterized by more intensive involvement of trans-splicing in RNA processing compared to other trematodes. CONCLUSION: All revealed peculiarities of structural organization of genomes are of extreme importance for a proper description of genes and their products in these parasitic species. This should be taken into account both in academic and applied research of epidemiologically important liver flukes. Further comparative genomics studies of liver flukes and non-carcinogenic flatworms allow for generation of well-grounded hypotheses on the mechanisms underlying development of cholangiocarcinoma associated with opisthorchiasis and clonorchiasis as well as species-specific mechanisms of these diseases.

The influence of obesity-related factors in the etiology of renal cell carcinoma-A mendelian randomization study.

BACKGROUND: Several obesity-related factors have been associated with renal cell carcinoma (RCC), but it is unclear which individual factors directly influence risk. We addressed this question using genetic markers as proxies for putative risk factors and evaluated their relation to RCC risk in a mendelian randomization (MR) framework. This methodology limits bias due to confounding and is not affected by reverse causation. METHODS AND FINDINGS: Genetic markers associated with obesity measures, blood pressure, lipids, type 2 diabetes, insulin, and glucose were initially identified as instrumental variables, and their association with RCC risk was subsequently evaluated in a genome-wide association study (GWAS) of 10,784 RCC patients and 20,406 control participants in a 2-sample MR framework. The effect on RCC risk was estimated by calculating odds ratios (ORSD) for a standard deviation (SD) increment in each risk factor. The MR analysis indicated that higher body mass index increases the risk of RCC (ORSD: 1.56, 95% confidence interval [CI] 1.44-1.70), with comparable results for waist-to-hip ratio (ORSD: 1.63, 95% CI 1.40-1.90) and body fat percentage (ORSD: 1.66, 95% CI 1.44-1.90). This analysis further indicated that higher fasting insulin (ORSD: 1.82, 95% CI 1.30-2.55) and diastolic blood pressure (DBP; ORSD: 1.28, 95% CI 1.11-1.47), but not systolic blood pressure (ORSD: 0.98, 95% CI 0.84-1.14), increase the risk for RCC. No association with RCC risk was seen for lipids, overall type 2 diabetes, or fasting glucose. CONCLUSIONS: This study provides novel evidence for an etiological role of insulin in RCC, as well as confirmatory evidence that obesity and DBP influence RCC risk.

Magnetospirillum kuznetsovii sp. nov., a novel magnetotactic bacterium isolated from a lake in the Moscow region.

Strain LBB-42T was isolated from sediment sampled at Lake Beloe Bordukovskoe, located in the Moscow region (Russia). Phylogenetic analyses based on 16S rRNA gene sequencing results assigned the strain to the genus Magnetospirillum. Major fatty acids were C16 : 1ω7c, C16 : 0 and C18 : 1 ω9/C18 : 1 ω7. Genome sequencing revealed a genome size of 4.40 Mbp and a G+C content of 63.4 mol%. The average nucleotide identity and digital DNA-DNA hybridization values suggested that strain LBB-42T represents a new species, for which we propose the name Magnetospirillum kuznetsovii sp. nov., with the type strain LBB-42T (=VKM B-3270T=KCTC 15749T).

Plastid Genomes of Carnivorous Plants Drosera rotundifolia and Nepenthes × ventrata Reveal Evolutionary Patterns Resembling Those Observed in Parasitic Plants.

Carnivorous plants have the ability to capture and digest small animals as a source of additional nutrients, which allows them to grow in nutrient-poor habitats. Here we report the complete sequences of the plastid genomes of two carnivorous plants of the order Caryophyllales, Drosera rotundifolia and Nepenthes × ventrata. The plastome of D. rotundifolia is repeat-rich and highly rearranged. It lacks NAD(P)H dehydrogenase genes, as well as ycf1 and ycf2 genes, and three essential tRNA genes. Intron losses are observed in some protein-coding and tRNA genes along with a pronounced reduction of RNA editing sites. Only six editing sites were identified by RNA-seq in D. rotundifolia plastid genome and at most conserved editing sites the conserved amino acids are already encoded at the DNA level. In contrast, the N. × ventrata plastome has a typical structure and gene content, except for pseudogenization of the ccsA gene. N. × ventrata and D. rotundifolia could represent different stages of evolution of the plastid genomes of carnivorous plants, resembling events observed in parasitic plants in the course of the switch from autotrophy to a heterotrophic lifestyle.

Between Lake Baikal and the Baltic Sea: genomic history of the gateway to Europe.

BACKGROUND: The history of human populations occupying the plains and mountain ridges separating Europe from Asia has been eventful, as these natural obstacles were crossed westward by multiple waves of Turkic and Uralic-speaking migrants as well as eastward by Europeans. Unfortunately, the material records of history of this region are not dense enough to reconstruct details of population history. These considerations stimulate growing interest to obtain a genetic picture of the demographic history of migrations and admixture in Northern Eurasia. RESULTS: We genotyped and analyzed 1076 individuals from 30 populations with geographical coverage spanning from Baltic Sea to Baikal Lake. Our dense sampling allowed us to describe in detail the population structure, provide insight into genomic history of numerous European and Asian populations, and significantly increase quantity of genetic data available for modern populations in region of North Eurasia. Our study doubles the amount of genome-wide profiles available for this region. We detected unusually high amount of shared identical-by-descent (IBD) genomic segments between several Siberian populations, such as Khanty and Ket, providing evidence of genetic relatedness across vast geographic distances and between speakers of different language families. Additionally, we observed excessive IBD sharing between Khanty and Bashkir, a group of Turkic speakers from Southern Urals region. While adding some weight to the "Finno-Ugric" origin of Bashkir, our studies highlighted that the Bashkir genepool lacks the main "core", being a multi-layered amalgamation of Turkic, Ugric, Finnish and Indo-European contributions, which points at intricacy of genetic interface between Turkic and Uralic populations. Comparison of the genetic structure of Siberian ethnicities and the geography of the region they inhabit point at existence of the "Great Siberian Vortex" directing genetic exchanges in populations across the Siberian part of Asia. Slavic speakers of Eastern Europe are, in general, very similar in their genetic composition. Ukrainians, Belarusians and Russians have almost identical proportions of Caucasus and Northern European components and have virtually no Asian influence. We capitalized on wide geographic span of our sampling to address intriguing question about the place of origin of Russian Starovers, an enigmatic Eastern Orthodox Old Believers religious group relocated to Siberia in seventeenth century. A comparative reAdmix analysis, complemented by IBD sharing, placed their roots in the region of the Northern European Plain, occupied by North Russians and Finno-Ugric Komi and Karelian people. Russians from Novosibirsk and Russian Starover exhibit ancestral proportions close to that of European Eastern Slavs, however, they also include between five to 10 % of Central Siberian ancestry, not present at this level in their European counterparts. CONCLUSIONS: Our project has patched the hole in the genetic map of Eurasia: we demonstrated complexity of genetic structure of Northern Eurasians, existence of East-West and North-South genetic gradients, and assessed different inputs of ancient populations into modern populations.

Genome sequence and analysis of the tuber crop potato.

Potato (Solanum tuberosum L.) is the world's most important non-grain food crop and is central to global food security. It is clonally propagated, highly heterozygous, autotetraploid, and suffers acute inbreeding depression. Here we use a homozygous doubled-monoploid potato clone to sequence and assemble 86% of the 844-megabase genome. We predict 39,031 protein-coding genes and present evidence for at least two genome duplication events indicative of a palaeopolyploid origin. As the first genome sequence of an asterid, the potato genome reveals 2,642 genes specific to this large angiosperm clade. We also sequenced a heterozygous diploid clone and show that gene presence/absence variants and other potentially deleterious mutations occur frequently and are a likely cause of inbreeding depression. Gene family expansion, tissue-specific expression and recruitment of genes to new pathways contributed to the evolution of tuber development. The potato genome sequence provides a platform for genetic improvement of this vital crop.

State of the Art of Chromosome 18-Centric HPP in 2016: Transcriptome and Proteome Profiling of Liver Tissue and HepG2 Cells.

A gene-centric approach was applied for a large-scale study of expression products of a single chromosome. Transcriptome profiling of liver tissue and HepG2 cell line was independently performed using two RNA-Seq platforms (SOLiD and Illumina) and also by Droplet Digital PCR (ddPCR) and quantitative RT-PCR. Proteome profiling was performed using shotgun LC-MS/MS as well as selected reaction monitoring with stable isotope-labeled standards (SRM/SIS) for liver tissue and HepG2 cells. On the basis of SRM/SIS measurements, protein copy numbers were estimated for the Chromosome 18 (Chr 18) encoded proteins in the selected types of biological material. These values were compared with expression levels of corresponding mRNA. As a result, we obtained information about 158 and 142 transcripts for HepG2 cell line and liver tissue, respectively. SRM/SIS measurements and shotgun LC-MS/MS allowed us to detect 91 Chr 18-encoded proteins in total, while an intersection between the HepG2 cell line and liver tissue proteomes was ∼66%. In total, there were 16 proteins specifically observed in HepG2 cell line, while 15 proteins were found solely in the liver tissue. Comparison between proteome and transcriptome revealed a poor correlation (R2 ≈ 0.1) between corresponding mRNA and protein expression levels. The SRM and shotgun data sets (obtained during 2015-2016) are available in PASSEL (PASS00697) and ProteomeExchange/PRIDE (PXD004407). All measurements were also uploaded into the in-house Chr 18 Knowledgebase at http://kb18.ru/protein/matrix/416126 .

Deep-sequence profiling of miRNAs and their target prediction in Monotropa hypopitys.

Myco-heterotroph Monotropa hypopitys is a widely spread perennial herb used to study symbiotic interactions and physiological mechanisms underlying the development of non-photosynthetic plant. Here, we performed, for the first time, transcriptome-wide characterization of M. hypopitys miRNA profile using high throughput Illumina sequencing. As a result of small RNA library sequencing and bioinformatic analysis, we identified 55 members belonging to 40 families of known miRNAs and 17 putative novel miRNAs unique for M. hypopitys. Computational screening revealed 206 potential mRNA targets for known miRNAs and 31 potential mRNA targets for novel miRNAs. The predicted target genes were described in Gene Ontology terms and were found to be involved in a broad range of metabolic and regulatory pathways. The identification of novel M. hypopitys-specific miRNAs, some with few target genes and low abundances, suggests their recent evolutionary origin and participation in highly specialized regulatory mechanisms fundamental for non-photosynthetic biology of M. hypopitys. This global analysis of miRNAs and their potential targets in M. hypopitys provides a framework for further investigation of miRNA role in the evolution and establishment of non-photosynthetic myco-heterotrophs.

Novel small molecule modulators of plant growth and development identified by high-content screening with plant pollen.

BACKGROUND: Small synthetic molecules provide valuable tools to agricultural biotechnology to circumvent the need for genetic engineering and provide unique benefits to modulate plant growth and development. RESULTS: We developed a method to explore molecular mechanisms of plant growth by high-throughput phenotypic screening of haploid populations of pollen cells. These cells rapidly germinate to develop pollen tubes. Compounds acting as growth inhibitors or stimulators of pollen tube growth are identified in a screen lasting not longer than 8 h high-lighting the potential broad applicability of this assay to prioritize chemicals for future mechanism focused investigations in plants. We identified 65 chemical compounds that influenced pollen development. We demonstrated the usefulness of the identified compounds as promotors or inhibitors of tobacco and Arabidopsis thaliana seed growth. When 7 days old seedlings were grown in the presence of these chemicals twenty two of these compounds caused a reduction in Arabidopsis root length in the range from 4.76 to 49.20 % when compared to controls grown in the absence of the chemicals. Two of the chemicals sharing structural homology with thiazolidines stimulated root growth and increased root length by 129.23 and 119.09 %, respectively. The pollen tube growth stimulating compound (S-02) belongs to benzazepin-type chemicals and increased Arabidopsis root length by 126.24 %. CONCLUSIONS: In this study we demonstrate the usefulness of plant pollen tube based assay for screening small chemical compound libraries for new biologically active compounds. The pollen tubes represent an ultra-rapid screening tool with which even large compound libraries can be analyzed in very short time intervals. The broadly applicable high-throughput protocol is suitable for automated phenotypic screening of germinating pollen resulting in combination with seed germination assays in identification of plant growth inhibitors and stimulators.

The loss of photosynthetic pathways in the plastid and nuclear genomes of the non-photosynthetic mycoheterotrophic eudicot Monotropa hypopitys.

BACKGROUND: Chloroplasts of most plants are responsible for photosynthesis and contain a conserved set of about 110 genes that encode components of housekeeping gene expression machinery and photosynthesis-related functions. Heterotrophic plants obtaining nutrients from other organisms and their plastid genomes represent model systems in which to study the effects of relaxed selective pressure on photosynthetic function. The most evident is a reduction in the size and gene content of the plastome, which correlates with the loss of genes encoding photosynthetic machinery which become unnecessary. Transition to a non-photosynthetic lifestyle is expected also to relax the selective pressure on photosynthetic machinery in the nuclear genome, however, the corresponding changes are less known. RESULTS: Here we report the complete sequence of the plastid genome of Monotropa hypopitys, an achlorophyllous obligately mycoheterotrophic plant belonging to the family Ericaceae. The plastome of M. hypopitys is greatly reduced in size (35,336 bp) and lacks the typical quadripartite structure with two single-copy regions and an inverted repeat. Only 45 genes remained presumably intact- those encoding ribosomal proteins, ribosomal and transfer RNA and housekeeping genes infA, matK, accD and clpP. The clpP and accD genes probably remain functional, although their sequences are highly diverged. The sets of genes for ribosomal protein and transfer RNA are incomplete relative to chloroplasts of a photosynthetic plant. Comparison of the plastid genomes of two subspecies-level isolates of M. hypopitys revealed major structural rearrangements associated with repeat-driven recombination and the presence of isolate-specific tRNA genes. Analysis of the M. hypopitys transcriptome by RNA-Seq showed the absence of expression of nuclear-encoded components of photosystem I and II reaction center proteins, components of cytochrome b6f complex, ATP synthase, ribulose bisphosphate carboxylase components, as well as chlorophyll from protoporphyrin IX biosynthesis pathway. CONCLUSIONS: With the complete loss of genes related to photosynthesis, NADH dehydrogenase, plastid-encoded RNA polymerase and ATP synthase, the M. hypopitys plastid genome is among the most functionally reduced ones characteristic of obligate non-photosynthetic parasitic species. Analysis of the M. hypopitys transcriptome revealed coordinated evolution of the nuclear and plastome genomes and the loss of photosynthesis-related functions in both genomes.

Understanding and Engineering Thermostability in DNA Ligase from Thermococcus sp. 1519.

The physical chemical principles underlying enzymatic thermostability are keys to understand the way evolution has shaped proteins to adapt to a broad range of temperatures. Understanding the molecular determinants at the basis of protein thermostability is also an important factor for engineering more thermoresistant enzymes to be used in the industrial setting, such as, for instance, DNA ligases, which are important for DNA replication and repair and have been long used in molecular biology and biotechnology. Here, we first address the origin of thermostability in the thermophilic DNA ligase from archaeon Thermococcus sp. 1519 and identify thermosensitive regions using molecular modeling and simulations. In addition, we predict mutations that can enhance thermostability of the enzyme through bioinformatics analyses. We show that thermosensitive regions of this enzyme are stabilized at higher temperatures by optimization of charged groups on the surface, and we predict that thermostability can be further increased by further optimization of the network among these charged groups. Engineering this DNA ligase by introducing selected mutations (i.e., A287K, G304D, S364I, and A387K) eventually produced a significant and additive increase in the half-life of the enzyme when compared to that of the wild type.

Specific features of telomerase RNA from Hansenula polymorpha.

Telomerase, a ribonucleoprotein, is responsible for the maintenance of eukaryotic genome integrity by replicating the ends of chromosomes. The core enzyme comprises the conserved protein TERT and an RNA subunit (TER) that, in contrast, displays large variations in size and structure. Here, we report the identification of the telomerase RNA from thermotolerant yeast Hansenula polymorpha (HpTER) and describe its structural features. We show further that the H. polymorpha telomerase reverse transcribes the template beyond the predicted boundary and adds a nontelomeric dT in vitro. Sequencing of the chromosomal ends revealed that this nucleotide is specifically present as a terminal nucleotide at the 3' end of telomeres. Mutational analysis of HpTER confirmed that the incorporation of dT functions to limit telomere length in this species.

The Geoglobus acetivorans genome: Fe(III) reduction, acetate utilization, autotrophic growth, and degradation of aromatic compounds in a hyperthermophilic archaeon.

Geoglobus acetivorans is a hyperthermophilic anaerobic euryarchaeon of the order Archaeoglobales isolated from deep-sea hydrothermal vents. A unique physiological feature of the members of the genus Geoglobus is their obligate dependence on Fe(III) reduction, which plays an important role in the geochemistry of hydrothermal systems. The features of this organism and its complete 1,860,815-bp genome sequence are described in this report. Genome analysis revealed pathways enabling oxidation of molecular hydrogen, proteinaceous substrates, fatty acids, aromatic compounds, n-alkanes, and organic acids, including acetate, through anaerobic respiration linked to Fe(III) reduction. Consistent with the inability of G. acetivorans to grow on carbohydrates, the modified Embden-Meyerhof pathway encoded by the genome is incomplete. Autotrophic CO2 fixation is enabled by the Wood-Ljungdahl pathway. Reduction of insoluble poorly crystalline Fe(III) oxide depends on the transfer of electrons from the quinone pool to multiheme c-type cytochromes exposed on the cell surface. Direct contact of the cells and Fe(III) oxide particles could be facilitated by pilus-like appendages. Genome analysis indicated the presence of metabolic pathways for anaerobic degradation of aromatic compounds and n-alkanes, although an ability of G. acetivorans to grow on these substrates was not observed in laboratory experiments. Overall, our results suggest that Geoglobus species could play an important role in microbial communities of deep-sea hydrothermal vents as lithoautotrophic producers. An additional role as decomposers would close the biogeochemical cycle of carbon through complete mineralization of various organic compounds via Fe(III) respiration.

Strain Improvement of Streptomyces xanthochromogenes RIA 1098 for Enhanced Pravastatin Production at High Compactin Concentrations.

Pravastatin is one of the most popular cholesterol-lowering drugs. Its industrial production represents a two-stage process including the microbial production of compactin and its further biocatalytic conversion to pravastatin. To increase a conversion rate, a higher compactin content in fermentation medium should be used; however, high compactin concentrations inhibit microbial growth. Therefore, the improvement of the compactin resistance of a producer still remains a relevant problem. A multi-step random UV mutagenesis of a Streptomyces xanthochromogenes strain RIA 1098 and the further selection of high-yield compactin-resistant mutants have resulted in a highly productive compactin-resistant strain S 33-1. After the fermentation medium improvement, the maximum bioconversion rate of this strain has reached 91 % at the daily compactin dose equal to 1 g/L and still remained high (83 %) even at the doubled dose (2 g/L). A 1-year study of the mutant strain stability has proved a stable inheritance of its characteristics that provides this strain to be very promising for the pravastatin-producing industry.

Chromosome 18 transcriptoproteome of liver tissue and HepG2 cells and targeted proteome mapping in depleted plasma: update 2013.

We report the results obtained in 2012-2013 by the Russian Consortium for the Chromosome-centric Human Proteome Project (C-HPP). The main scope of this work was the transcriptome profiling of genes on human chromosome 18 (Chr 18), as well as their encoded proteome, from three types of biomaterials: liver tissue, the hepatocellular carcinoma-derived cell line HepG2, and blood plasma. The transcriptome profiling for liver tissue was independently performed using two RNaseq platforms (SOLiD and Illumina) and also by droplet digital PCR (ddPCR) and quantitative RT-PCR. The proteome profiling of Chr 18 was accomplished by quantitatively measuring protein copy numbers in the three types of biomaterial (the lowest protein concentration measured was 10(-13) M) using selected reaction monitoring (SRM). In total, protein copy numbers were estimated for 228 master proteins, including quantitative data on 164 proteins in plasma, 171 in the HepG2 cell line, and 186 in liver tissue. Most proteins were present in plasma at 10(8) copies/µL, while the median abundance was 10(4) and 10(5) protein copies per cell in HepG2 cells and liver tissue, respectively. In summary, for liver tissue and HepG2 cells a "transcriptoproteome" was produced that reflects the relationship between transcript and protein copy numbers of the genes on Chr 18. The quantitative data acquired by RNaseq, PCR, and SRM were uploaded into the "Update_2013" data set of our knowledgebase (www.kb18.ru) and investigated for linear correlations.

Improved elongation factor-1 alpha-based vectors for stable high-level expression of heterologous proteins in Chinese hamster ovary cells.

BACKGROUND: Establishing highly productive clonal cell lines with constant productivity over 2-3 months of continuous culture remains a tedious task requiring the screening of tens of thousands of clonal colonies. In addition, long-term cultivation of many candidate lines derived in the absence of drug selection pressure is necessary. Expression vectors based on the elongation factor-1 alpha (EEF1A) gene and the dihydrofolate reductase (DHFR) selection marker (with separate promoters) can be used to obtain highly productive populations of stably transfected cells in the selection medium, but they have not been tested for their ability to support target gene amplification under gradually increasing methotrexate pressure. RESULTS: We have modified EEF1A-based vectors by linking the DHFR selection marker to the target gene in the bicistronic RNA, shortening the overall plasmid size, and adding an Epstein-Barr virus terminal repeat fragment (EBVTR) element. Presence of the EBVTR element increased the rate of stable transfection by the plasmid by 24 times that of the EBVTR-minus control and improved the rate of methotrexate-driven gene amplification. The mean expression level of the enhanced green fluorescent protein (eGFP) used herein as a model protein, increased up to eight-fold using a single round of amplification in the case of adherent colonies formation and up to 4.5-fold in the case of suspension polyclonal cultures. Several eGFP-expressing cell populations produced using vectors with antibiotic resistance markers instead of the DHFR marker were compared with each other. Stable transfection of Chinese hamster ovary (CHO) DG44 cells by the p1.2-Hygro-eGFP plasmid (containing a hygromycin resistance marker) generated highest eGFP expression levels of up to 8.9% of the total cytoplasmic protein, with less than 5% of the cell population being eGFP-negative. CONCLUSIONS: The p1.1 vector was very effective for stable transfection of CHO cells and capable of rapid MTX-driven target gene amplification, while p1.2-Hygro achieved similar eGFP expression levels as p1.1. The set of vectors we have developed should speed-up the process of generating highly productive clonal cell lines while substantially decreasing the associated experimental effort.

Analysis of the complete genome of Fervidococcus fontis confirms the distinct phylogenetic position of the order Fervidicoccales and suggests its environmental function.

The complete genome of the obligately anaerobic crenarchaeote Fervidicoccus fontis Kam940(T), a terrestrial hot spring inhabitant with a growth optimum of 65-70 °C, has been sequenced and analyzed. The small 1.3-Mb genome encodes several extracellular proteases and no other extracellular hydrolases. No complete pathways of carbohydrate catabolism were found. Genes coding for enzymes necessary for amino acid transamination and further oxidative decarboxylation are present. The genome encodes no mechanisms of acyl-CoA and acetyl-CoA oxidation. Two [NiFe]-hydrogenases are encoded: a membrane-bound energy-converting hydrogenase and a cytoplasmic one. The ATP-synthase is H(+)-dependent as inferred from the amino acid sequence of the membrane rotor subunit. On the whole, genome analysis shows F. fontis to be a peptidolytic heterotroph with a restricted biosynthetic potential, which is in accordance with its phenotypic properties. The analysis of phylogenetic markers and of the distribution of best blastp hits of F. fontis proteins in the available genomes of Crenarchaeota supports distinct phylogenetic position of the order Fervidicoccales as a separate lineage adjoining the heterogeneous order Desulfurococcales. In addition, certain F. fontis genomic features correlate with its adaptation to temperatures of 60-80 °C, which are lower than temperatures preferred by Desulfurococcales.

Genome sequence and analysis of methylotrophic yeast Hansenula polymorpha DL1.

BACKGROUND: Hansenula polymorpha DL1 is a methylotrophic yeast, widely used in fundamental studies of methanol metabolism, peroxisome biogenesis and function, and also as a microbial cell factory for production of recombinant proteins and metabolic engineering towards the goal of high temperature ethanol production. RESULTS: We have sequenced the 9 Mbp H. polymorpha DL1 genome and performed whole-genome analysis for the H. polymorpha transcriptome obtained from both methanol- and glucose-grown cells. RNA-seq analysis revealed the complex and dynamic character of the H. polymorpha transcriptome under the two studied conditions, identified abundant and highly unregulated expression of 40% of the genome in methanol grown cells, and revealed alternative splicing events. We have identified subtelomerically biased protein families in H. polymorpha, clusters of LTR elements at G + C-poor chromosomal loci in the middle of each of the seven H. polymorpha chromosomes, and established the evolutionary position of H. polymorpha DL1 within a separate yeast clade together with the methylotrophic yeast Pichia pastoris and the non-methylotrophic yeast Dekkera bruxellensis. Intergenome comparisons uncovered extensive gene order reshuffling between the three yeast genomes. Phylogenetic analyses enabled us to reveal patterns of evolution of methylotrophy in yeasts and filamentous fungi. CONCLUSIONS: Our results open new opportunities for in-depth understanding of many aspects of H. polymorpha life cycle, physiology and metabolism as well as genome evolution in methylotrophic yeasts and may lead to novel improvements toward the application of H. polymorpha DL-1 as a microbial cell factory.

Adult Opisthorchis felineus major protein fractions deduced from transcripts: comparison with liver flukes Opisthorchis viverrini and Clonorchis sinensis.

The epidemiologically important liver flukes Opisthorchis felineus, Opisthorchis viverrini, and Clonorchis sinensis are of interest to health professionals, epidemiologists, pharmacologists, and molecular biologists. Recently the transcriptomes of the latter two species were intensively investigated. However our knowledge on molecular biology of O. felineus is scarce. We report the first results of the O. felineus transcriptome analysis. We isolated and annotated a total of 2560 expressed sequence tag (EST) sequences from adult O. felineus (deposited within the database of expressed sequence tags (dbEST), under accession numbers GenBank: JK624271-JK626790, JK006511-JK006547, JK649790-JK649792). Clustering and analysis resulted in the detection of 267 contigs. Of the protein sequences deduced from these, 82% had homologs in the NCBI (nr) protein database and 63% contained conserved domains, allowing the functions to be interpreted using the Gene Ontology terms. Comprehensive analysis of Opisthorchiidae- and Trematoda-specific substitutions within amino acid sequences deduced for the proteins myoglobin, vitelline precursor protein, cathepsin F, and 28kDa glutathione transferase was carried out. The gene set of the 32 ribosomal proteins for the three Opisthorchiidae species with the addition of available Schistosoma and Fasciola orthologs was created and is provided in the supplementary. The orthologous gene set created was used for inferring phylogeny within the Trematoda with special attention to interrelations within the Opisthorchiidae. The phylogenetic analysis revealed a closer relationship between C. sinensis and O. viverrini and some divergence of O. felineus from either O. viverrini or C. sinensis.