Robert Gromadka

Paramecium genome survey : a pilot project

A consortium of laboratories undertook a pilot sequencing project to gain insight into the genome of Paramecium. Plasmid-end sequencing of DNA fragments from the somatic nucleus together with similarity searches identified 722 potential protein-coding genes. High gene density and uniform small intron size make random sequencing of somatic chromosomes a cost-effective strategy for gene discovery in this organism.

Sequencing the Potato Genome: Outline and First Results to Come from the Elucidation of the Sequence of the World's Third Most Important Food Crop

Potato is a member of the Solanaceae, a plant family that includes several other economically important species, such as tomato, eggplant, petunia, tobacco and pepper. The Potato Genome Sequencing Consortium (PGSC) aims to elucidate the complete genome sequence of potato, the third most important food crop in the world. The PGSC is a collaboration between 13 research groups from China, India, Poland, Russia, the Netherlands, Ireland, Argentina, Brazil, Chile, Peru, USA, New Zealand and the UK. The potato genome consists of 12 chromosomes and has a (haploid) length of approximately 840 million base pairs, making it a medium-sized plant genome. The sequencing project builds on a diploid potato genomic bacterial artificial chromosome (BAC) clone library of 78000 clones, which has been fingerprinted and aligned into ~7000 physical map contigs. In addition, the BAC-ends have been sequenced and are publicly available. Approximately 30000 BACs are anchored to the Ultra High Density genetic map of potato, composed of 10000 unique AFLPTM markers. From this integrated genetic-physical map, between 50 to 150 seed BACs have currently been identified for every chromosome. Fluorescent in situ hybridization experiments on selected BAC clones confirm these anchor points. The seed clones provide the starting point for a BAC-by-BAC sequencing strategy. This strategy is being complemented by whole genome shotgun sequencing approaches using both 454 GS FLX and Illumina GA2 instruments. Assembly and annotation of the sequence data will be performed using publicly available and tailor-made tools. The availability of the annotated data will help to characterize germplasm collections based on allelic variance and to assist potato breeders to more fully exploit the genetic potential of potato.ResumenLa papa es un miembro de las Solanaceae, una familia de plantas que incluye varias otras especies económicamente importantes, tales como tomate, berenjena, petunia, tabaco y ají o chili. El consorcio de secuenciación del genoma de la papa (PGSC) tiene por objeto dilucidar la secuencia completa del genoma de la papa, el tercer cultivo alimentario más importante del mundo. El PGSC es una colaboración entre 13 grupos de investigación procedentes de China, India, Polonia, Rusia, los Países Bajos, Irlanda, Argentina, Brasil, Chile, Perú, EE.UU., Nueva Zelanda y el Reino Unido. El genoma de la papa consiste de 12 cromosomas y tiene una longitud (haploide) de aproximadamente 840 millones de pares de bases, por lo que es una planta con un genoma de tamaño mediano. El proyecto de secuenciación se basa en una biblioteca de 78000 clones de cromosoma artificial bacteriano genomico de papa diploide (BAC), del que se ha obtenido la huella genética y alineado en 7000 ~ contigs de mapa físico. Además, los extremos terminales BAC se han secuenciado y están a disposición del público. Aproximadamente 30000 BACS están anclados al mapa genético de ultra alta densidad de la papa, compuesto de 10000 marcadores AFLPTM únicos. De esta mapa genético-físico integrado, entre 50 a 150 semillas BACs han sido identificadas para cada cromosoma. Experimentos de hibridación in situ fluorescente en clones BAC selectos confirman estos puntos de anclaje. La clones semilla proveen el punto de partida para la estrategia de secuenciación de BAC a BAC. Esta estrategia se complementa con los enfoques de secuenciación escopeta del genoma completo usando los instrumentos 454 GS FLX e Illumina GA2. El ensamblaje y anotación de los datos de la secuencia será realizados mediante herramientas publicas disponibles y hechas a la medida. La disponibilidad de los datos anotados ayudarán a caracterizar las colecciones de germoplasma basándose en variación alélica y ayudará a los fitomejoradores de papa a explotar más plenamente el potencial genético de la papa.

High Coding Density on the Largest Paramecium tetraurelia Somatic Chromosome

Paramecium, like other ciliates, remodels its entire germline genome at each sexual generation to produce a somatic genome stripped of transposons and other multicopy elements. The germline chromosomes are fragmented by a DNA elimination process that targets heterochromatin to give a reproducible set of some 200 linear molecules 50 kb to 1 Mb in size. These chromosomes are maintained at a ploidy of 800n in the somatic macronucleus and assure all gene expression. We isolated and sequenced the largest megabase somatic chromosome in order to explore its organization and gene content. The AT-rich (72%) chromosome is compact, with very small introns (average size 25 nt), short intergenic regions (median size 202 nt), and a coding density of at least 74%, higher than that reported for budding yeast (70%) or any other free-living eukaryote. Similarity to known proteins could be detected for 57% of the 460 potential protein coding genes. Thirty-two of the proteins are shared with vertebrates but absent from yeast, consistent with the morphogenetic complexity of Paramecium, a long-standing model for differentiated functions shared with metazoans but often absent from simpler eukaryotes. Extrapolation to the whole genome suggests that Paramecium has at least 30,000 genes.

Random Sequencing of Paramecium Somatic DNA

ABSTRACT We report a random survey of 1 to 2% of the somatic genome of the free-living ciliate Paramecium tetraurelia by single-run sequencing of the ends of plasmid inserts. As in all ciliates, the germ line genome of Paramecium (100 to 200 Mb) is reproducibly rearranged at each sexual cycle to produce a somatic genome of expressed or potentially expressed genes, stripped of repeated sequences, transposons, and AT-rich unique sequence elements limited to the germ line. We found the somatic genome to be compact (>68% coding, estimated from the sequence of several complete library inserts) and to feature uniformly small introns (18 to 35 nucleotides). This facilitated gene discovery: 722 open reading frames (ORFs) were identified by similarity with known proteins, and 119 novel ORFs were tentatively identified by internal comparison of the data set. We determined the phylogenetic position of Paramecium with respect to eukaryotes whose genomes have been sequenced by the distance matrix neighbor-joining method by using random combined protein data from the project. The unrooted tree obtained is very robust and in excellent agreement with accepted topology, providing strong support for the quality and consistency of the data set. Our study demonstrates that a random survey of the somatic genome of Paramecium is a good strategy for gene discovery in this organism.

Two different species of Euglena, E. geniculata and E. myxocylindracea (euglenophyceae), are virtually genetically and morphologically identical

We investigated the similarity of a single Euglena myxocylindracea strain, isolated originally by Bold and MacEntee, to several Euglena geniculata strains on both morphological and DNA levels. We found the three DNA stretches, consisting of fragments coding for the parts of cytoplasmic and chloroplast small subunit rRNA, and the internal transcribed spacer (ITS2) of cytoplasmic rDNA, with the combined length of 4332 nucleotides, are identical in E. myxocylindracea and E. geniculata, strain SAG 1224‐4b. Morphological differences between E. myxocylindracea and any E. geniculata strain examined were well within the range of E. geniculata variability as well. The only difference behind the distinction of E. myxocylindracea from E. geniculata is the presence of the second chloroplast in the latter. However, we were able to induce the appearance of the second chloroplast in the cells of E. myxocylindracea and its disappearance in the cells of E. geniculata by changing the composition of the culture media. We therefore conclude that E. myxocylindracea Bold and MacEntee should be regarded as an environmental form of E. geniculata Dujardin. For the first time the morphology of E. geniculata chloroplasts was shown as revealed by confocal laser microscopy.

Disruption of six novel yeast genes located on chromosome II reveals one gene essential for vegetative growth and two required for sporulation and conferring hypersensitivity to various chemicals.

A PCR‐based method for targeted gene deletion by kanMX4 module was used to construct complete deletion mutants of six individual open reading frames from chromosome II: YBR128c, YBR131w, YBR133c, YBR137w, YBR138c and YBR142w. The ORFs were deleted in two diploid strains, FY1679 and W303. Sporulation and tetrad analysis revealed that only one ORF, YBR142w, encoding a putative DEAD‐box RNA helicase, is an essential gene. A systematic phenotypic analysis of the deleted mutants was carried out. Homozygous diploids ybr128cΔ/ybr128cΔ and ybr131wΔ/ybr131wΔ did not sporulate. The ybr131cΔ mutant whether haploid or homozygous diploid, in addition displayed an increased sensitivity to Caffeine, Calcium and Zinc, and to emphasize this phenotype we named the gene CCZ1. ORF YBR133c was independently reported by others as Histone Synthetic Lethal (HSL7) (Ma et al., 1996). We found that the aberrant morphology characteristic for ybr133cΔ (hsl7Δ) cells was observed in W303 but not in FY1679 genetic background. Furthermore, we observed that deletion of YBR133c had a pleiotropic effect under a wide range of conditions, including increased sensitivity to calcium, caffeine, calcofluor white, vanadate and verapamil. The effects of the deletion were reinforced in W303 background. We found no phenotypic effects of the two remaining deletions, ybr137wΔ and ybr138cΔ. Copyright

Functional study of genes essential for autogamy and nuclear reorganization in Paramecium.

ABSTRACT Like all ciliates, Paramecium tetraurelia is a unicellular eukaryote that harbors two kinds of nuclei within its cytoplasm. At each sexual cycle, a new somatic macronucleus (MAC) develops from the germ line micronucleus (MIC) through a sequence of complex events, which includes meiosis, karyogamy, and assembly of the MAC genome from MIC sequences. The latter process involves developmentally programmed genome rearrangements controlled by noncoding RNAs and a specialized RNA interference machinery. We describe our first attempts to identify genes and biological processes that contribute to the progression of the sexual cycle. Given the high percentage of unknown genes annotated in the P. tetraurelia genome, we applied a global strategy to monitor gene expression profiles during autogamy, a self-fertilization process. We focused this pilot study on the genes carried by the largest somatic chromosome and designed dedicated DNA arrays covering 484 genes from this chromosome (1.2% of all genes annotated in the genome). Transcriptome analysis revealed four major patterns of gene expression, including two successive waves of gene induction. Functional analysis of 15 upregulated genes revealed four that are essential for vegetative growth, one of which is involved in the maintenance of MAC integrity and another in cell division or membrane trafficking. Two additional genes, encoding a MIC-specific protein and a putative RNA helicase localizing to the old and then to the new MAC, are specifically required during sexual processes. Our work provides a proof of principle that genes essential for meiosis and nuclear reorganization can be uncovered following genome-wide transcriptome analysis.

Evidence of adaptation, niche separation and microevolution within the genus Polaromonas on Arctic and Antarctic glacial surfaces

Polaromonas is one of the most abundant genera found on glacier surfaces, yet its ecology remains poorly described. Investigations made to date point towards a uniform distribution of Polaromonas phylotypes across the globe. We compared 43 Polaromonas isolates obtained from surfaces of Arctic and Antarctic glaciers to address this issue. 16S rRNA gene sequences, intergenic transcribed spacers (ITS) and metabolic fingerprinting showed great differences between hemispheres but also between neighboring glaciers. Phylogenetic distance between Arctic and Antarctic isolates indicated separate species. The Arctic group clustered similarly, when constructing dendrograms based on 16S rRNA gene and ITS sequences, as well as metabolic traits. The Antarctic strains, although almost identical considering 16S rRNA genes, diverged into 2 groups based on the ITS sequences and metabolic traits, suggesting recent niche separation. Certain phenotypic traits pointed towards cell adaptation to specific conditions on a particular glacier, like varying pH levels. Collected data suggest, that seeding of glacial surfaces with Polaromonas cells transported by various means, is of greater efficiency on local than global scales. Selection mechanisms present of glacial surfaces reduce the deposited Polaromonas diversity, causing subsequent adaptation to prevailing environmental conditions. Furthermore, interactions with other supraglacial microbiota, like algae cells may drive postselectional niche separation and microevolution within the Polaromonas genus.

TFIIS-Dependent Non-coding Transcription Regulates Developmental Genome Rearrangements

Because of their nuclear dimorphism, ciliates provide a unique opportunity to study the role of non-coding RNAs (ncRNAs) in the communication between germline and somatic lineages. In these unicellular eukaryotes, a new somatic nucleus develops at each sexual cycle from a copy of the zygotic (germline) nucleus, while the old somatic nucleus degenerates. In the ciliate Paramecium tetraurelia, the genome is massively rearranged during this process through the reproducible elimination of repeated sequences and the precise excision of over 45,000 short, single-copy Internal Eliminated Sequences (IESs). Different types of ncRNAs resulting from genome-wide transcription were shown to be involved in the epigenetic regulation of genome rearrangements. To understand how ncRNAs are produced from the entire genome, we have focused on a homolog of the TFIIS elongation factor, which regulates RNA polymerase II transcriptional pausing. Six TFIIS-paralogs, representing four distinct families, can be found in P. tetraurelia genome. Using RNA interference, we showed that TFIIS4, which encodes a development-specific TFIIS protein, is essential for the formation of a functional somatic genome. Molecular analyses and high-throughput DNA sequencing upon TFIIS4 RNAi demonstrated that TFIIS4 is involved in all kinds of genome rearrangements, including excision of ~48% of IESs. Localization of a GFP-TFIIS4 fusion revealed that TFIIS4 appears specifically in the new somatic nucleus at an early developmental stage, before IES excision. RT-PCR experiments showed that TFIIS4 is necessary for the synthesis of IES-containing non-coding transcripts. We propose that these IES+ transcripts originate from the developing somatic nucleus and serve as pairing substrates for germline-specific short RNAs that target elimination of their homologous sequences. Our study, therefore, connects the onset of zygotic non coding transcription to the control of genome plasticity in Paramecium, and establishes for the first time a specific role of TFIIS in non-coding transcription in eukaryotes.

Enrichment of Cryoconite Hole Anaerobes: Implications for the Subglacial Microbiome

Glaciers have recently been recognized as ecosystems comprised of several distinct habitats: a sunlit and oxygenated glacial surface, glacial ice, and a dark, mostly anoxic glacial bed. Surface meltwaters annually flood the subglacial sediments by means of drainage channels. Glacial surfaces host aquatic microhabitats called cryoconite holes, regarded as “hot spots” of microbial abundance and activity, largely contributing to the meltwaters’ bacterial diversity. This study presents an investigation of cryoconite hole anaerobes and discusses their possible impact on subglacial microbial communities, combining 16S rRNA gene fragment amplicon sequencing and the traditional enrichment culture technique. Cryoconite hole sediment harbored bacteria belonging mainly to the Proteobacteria (21%), Bacteroidetes (16%), Actinobacteria (14%), and Planctomycetes (6%) phyla. An 8-week incubation of those sediments in Postgate C medium for sulfate reducers in airtight bottles, emulating subglacial conditions, eliminated a great majority of dominant taxa, leading to enrichment of the Firmicutes (62%), Proteobacteria (14%), and Bacteroidetes (13%), which consisted of anaerobic genera like Clostridium, Psychrosinus, Paludibacter, and Acetobacterium. Enrichment of Pseudomonas spp. also occurred, suggesting it played a role as a dominant oxygen scavenger, providing a possible scenario for anaerobic niche establishment in subglacial habitats. To our knowledge, this is the first paper to provide insight into the diversity of the anaerobic part of the cryoconite hole microbial community and its potential to contribute to matter turnover in anoxic, subglacial sites.