K. G. Skryabin

Conserved and variable elements in RNA genomes of potexviruses

The nucleotide sequences of genomic RNAs and predicted amino acid sequences of two strains of potato virus X and white clover mosaic potexvirus were compared to each other, and the proteins of different plus‐RNA‐containing plant viruses. The predicted non‐virion proteins of potexviruses have direct sequence homology and common structural peculiarities with those of several ‘Sindbis‐like’ plant viruses. The most conserved amino acid sequences were found to be located in the polypeptide encoded by the long 5′‐proximal open reading frame (ORF1). The putative polypeptide encoded by the ORF2 starting beyond the ORF1 stop codon is clearly related to the presumptive NTP‐binding domain of the ORF1‐coded polypeptide. These results suggest possible functions for all of the potexvirus proteins and also indicate that potexviruses have a genome organization which is considerably different from that of other plant viruses.

Identification and Characterization of Four Chrysanthemum MADS-Box Genes, Belonging to the APETALA1/FRUITFULL and SEPALLATA3 Subfamilies

Four full-length MADS-box cDNAs from chrysanthemum, designated Chrysanthemum Dendrathema grandiflorum MADS (CDM) 8, CDM41, CDM111, and CDM44, have been isolated and further functionally characterized. Protein sequence alignment and expression patterns of the corresponding genes suggest that CDM8 and CDM41 belong to the FRUITFULL (FUL) clade, CDM111 is a member of the APETALA1 (AP1) subfamily, and CDM44 is a member of the SEPALLATA3 (SEP3) subfamily of MADS-box transcription factors. Overexpression of CDM111 in Arabidopsis plants resulted in an aberrant phenotype that is reminiscent of the phenotype obtained by ectopic expression of the AP1 gene. In addition, CDM111 was able to partially complement the ap1-1 mutant from Arabidopsis, illustrating that CDM111 is the functional equivalent to AP1. Yeast two- and three-hybrid studies were performed to investigate the potential protein interactions and complexes in which these chrysanthemum MADS-box proteins are involved. Based on these studies, we conclude that CDM44 is most likely the SEP3 functional equivalent, because the CDM44 protein interacts with CDM proteins of the AP1/FUL and AG subfamilies, and as a higher order complex with the heterodimer between the presumed B-type CDM proteins.

Metabolic Versatility and Indigenous Origin of the Archaeon Thermococcus sibiricus, Isolated from a Siberian Oil Reservoir, as Revealed by Genome Analysis

ABSTRACT Thermococcus species are widely distributed in terrestrial and marine hydrothermal areas, as well as in deep subsurface oil reservoirs. Thermococcus sibiricus is a hyperthermophilic anaerobic archaeon isolated from a well of the never flooded oil-bearing Jurassic horizon of a high-temperature oil reservoir. To obtain insight into the genome of an archaeon inhabiting the oil reservoir, we have determined and annotated the complete 1,845,800-base genome of T. sibiricus. A total of 2,061 protein-coding genes have been identified, 387 of which are absent in other members of the order Thermococcales. Physiological features and genomic data reveal numerous hydrolytic enzymes (e.g., cellulolytic enzymes, agarase, laminarinase, and lipases) and metabolic pathways, support the proposal of the indigenous origin of T. sibiricus in the oil reservoir, and explain its survival over geologic time and its proliferation in this habitat. Indeed, in addition to proteinaceous compounds known previously to be present in oil reservoirs at limiting concentrations, its growth was stimulated by cellulose, agarose, and triacylglycerides, as well as by alkanes. Two polysaccharide degradation loci were probably acquired by T. sibiricus from thermophilic bacteria following lateral gene transfer events. The first, a “saccharolytic gene island” absent in the genomes of other members of the order Thermococcales, contains the complete set of genes responsible for the hydrolysis of cellulose and β-linked polysaccharides. The second harbors genes for maltose and trehalose degradation. Considering that agarose and laminarin are components of algae, the encoded enzymes and the substrate spectrum of T. sibiricus indicate the ability to metabolize the buried organic matter from the original oceanic sediment.

Variation in genomic landscape of clear cell renal cell carcinoma across Europe

The incidence of renal cell carcinoma (RCC) is increasing worldwide, and its prevalence is particularly high in some parts of Central Europe. Here we undertake whole-genome and transcriptome sequencing of clear cell RCC (ccRCC), the most common form of the disease, in patients from four different European countries with contrasting disease incidence to explore the underlying genomic architecture of RCC. Our findings support previous reports on frequent aberrations in the epigenetic machinery and PI3K/mTOR signalling, and uncover novel pathways and genes affected by recurrent mutations and abnormal transcriptome patterns including focal adhesion, components of extracellular matrix (ECM) and genes encoding FAT cadherins. Furthermore, a large majority of patients from Romania have an unexpected high frequency of A:T>T:A transversions, consistent with exposure to aristolochic acid (AA). These results show that the processes underlying ccRCC tumorigenesis may vary in different populations and suggest that AA may be an important ccRCC carcinogen in Romania, a finding with major public health implications.

A novel type of crystallin in the frog eye lens. 35-kDa polypeptide is not homologous to any of the major classes of lens crystallins.

The nucleotide sequence of a cloned DNA coding for the 35‐kDa polypeptide of the eye lens of the frog Rana temporaria has been determined. The sequence without connectors and poly(A) tract is 889 nucleotides in length and shows no homology with sequences coding for other classes of crystallins; α‐, β‐, γ‐ or δ‐crystallins. The sequence contains one reading frame 675 nucleotides in length, an apparently intact 3′‐non‐translated region with the polyadenylation signal sequence and a poly(A) tract; the 5′‐non‐ translated region is lost along with part of the coding region; this accounts for about 1/4 of the total mRNA length. The secondary structure prediction according to the Ptitsin‐Finkelstein method shows the presence of predominantly β‐strands with only a few α‐helical regions. We conclude that the 35‐kDa polypeptide from the frog eye lens belongs to a new class of eye lens crystallins for which we propose the name ϵ‐crystallin.

Microbial community structure in methane hydrate-bearing sediments of freshwater Lake Baikal.

Gas hydrates in marine sediments have been known for many years but recently hydrates were found in the sediments of Lake Baikal, the largest freshwater basin in the world. Marine gas hydrates are associated with complex microbial communities involved in methanogenesis, methane oxidation, sulfate reduction and other biotransformations. However, the contribution of microorganisms to the formation of gas hydrates remains poorly understood. We examined the microbial communities in the hydrate-bearing sediments and water column of Lake Baikal using pyrosequencing of 16S rRNA genes. Aerobic methanotrophic bacteria dominated the water sample collected at the lake floor in the hydrate-bearing site. The shallow sediments were dominated by Archaea. Methanogens of the orders Methanomicrobiales and Methanosarcinales were abundant, whereas representatives of archaeal lineages known to perform anaerobic oxidation of methane, as well as sulfate-reducing bacteria, were not found. Affiliation of archaea to methanogenic rather than methane-oxidizing lineages was supported by analysis of the sequences of the methyl coenzyme M reductase gene. The deeper sediments located at 85-90 cm depth close to the hydrate were dominated by Bacteria, mostly assigned to Chloroflexi, candidate division JS1 and Caldiserica. Overall, our results are consistent with the biological origin of methane hydrates in Lake Baikal.

Role of the leader sequence in tobacco pectin methylesterase secretion

We report that unprocessed tobacco pectin methylesterase (PME) contains N‐terminal pro‐sequence including the transmembrane (TM) domain and spacer segment preceding the mature PME. The mature portion of PME was replaced by green fluorescent protein (GFP) gene and various deletion mutants of pro‐sequence fused to GFP were cloned into binary vectors and agroinjected in Nicotiana benthamiana leaves. The PME pro‐sequence delivered GFP to the cell wall (CW). We showed that a transient binding of PME TM domain to endoplasmic reticulum membranes occurs upon its transport to CW. The CW targeting was abolished by various deletions in the TM domain, i.e., anchor domain was essential for secretion of GFP to CW. By contrast, even entire deletion of the spacer segment had no influence on GFP targeting.

Genome sequence and analysis of methylotrophic yeast Hansenula polymorpha DL1

BackgroundHansenula 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.ResultsWe 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.ConclusionsOur 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.

A novel function for a ubiquitous plant enzyme pectin methylesterase: The enhancer of RNA silencing

Co‐agroinjection of Nicotiana benthamiana leaves with the pectin methylesterase (proPME) gene and the TMV:GFP vector resulted in a stimulation of virus‐induced RNA silencing (inhibition of GFP production, virus RNA degradation, stimulation of siRNAs production). Conversely, co‐expression of TMV:GFP with either antisense PME construct or with enzymatically inactive proPME restored synthesis of viral RNA. Furthermore, expression of proPME enhanced the GFP transgene‐induced gene silencing accompanied by relocation of the DCL1 protein from nucleus to the cytoplasm and activation of siRNAs and miRNAs production. It was hypothesized that DCL1 relocated to the cytoplasm may use as substrates both miRNA precursor and viral RNA. The capacity for enhancing the RNA silencing is a novel function for the polyfunctional PME.

Complete Genome Sequence of the Anaerobic, Protein-Degrading Hyperthermophilic Crenarchaeon Desulfurococcus kamchatkensis

Desulfurococcus kamchatkensis is an anaerobic organotrophic hyperthermophilic crenarchaeon isolated from a terrestrial hot spring. Its genome consists of a single circular chromosome of 1,365,223 bp with no extrachromosomal elements. A total of 1,474 protein-encoding genes were annotated, among which 205 are exclusive for D. kamchatkensis. The search for a replication origin site revealed a single region coinciding with a global extreme of the nucleotide composition disparity curve and containing a set of crenarchaeon-type origin recognition boxes. Unlike in most archaea, two genes encoding homologs of the eukaryotic initiator proteins Orc1 and Cdc6 are located distantly from this site. A number of mobile elements are present in the genome, including seven transposons representing IS607 and IS200/IS605 families and multiple copies of miniature inverted repeat transposable elements. Two large clusters of regularly interspaced repeats are present; none of the spacer sequences matches known archaeal extrachromosomal elements, except one spacer matches the sequence of a resident gene of D. kamchatkensis. Many of the predicted metabolic enzymes are associated with the fermentation of peptides and sugars, including more than 30 peptidases with diverse specificities, a number of polysaccharide degradation enzymes, and many transporters. Consistently, the genome encodes both enzymes of the modified Embden-Meyerhof pathway of glucose oxidation and a set of enzymes needed for gluconeogenesis. The genome structure and content reflect the organisms nutritionally diverse, competitive natural environment, which is periodically invaded by viruses and other mobile elements.