Rama K. Singh

The complete genome of the crenarchaeon Sulfolobus solfataricus P2

The genome of the crenarchaeon Sulfolobus solfataricus P2 contains 2,992,245 bp on a single chromosome and encodes 2,977 proteins and many RNAs. One-third of the encoded proteins have no detectable homologs in other sequenced genomes. Moreover, 40% appear to be archaeal-specific, and only 12% and 2.3% are shared exclusively with bacteria and eukarya, respectively. The genome shows a high level of plasticity with 200 diverse insertion sequence elements, many putative nonautonomous mobile elements, and evidence of integrase-mediated insertion events. There are also long clusters of regularly spaced tandem repeats. Different transfer systems are used for the uptake of inorganic and organic solutes, and a wealth of intracellular and extracellular proteases, sugar, and sulfur metabolizing enzymes are encoded, as well as enzymes of the central metabolic pathways and motility proteins. The major metabolic electron carrier is not NADH as in bacteria and eukarya but probably ferredoxin. The essential components required for DNA replication, DNA repair and recombination, the cell cycle, transcriptional initiation and translation, but not DNA folding, show a strong eukaryal character with many archaeal-specific features. The results illustrate major differences between crenarchaea and euryarchaea, especially for their DNA replication mechanism and cell cycle processes and their translational apparatus.

Organizational characteristics and information content of an archaeal genome: 156 kb of sequence from Sulfolobus solfataricus P2.

We have initiated a project to sequence the 3Mbp genome of the thermoacidophilic archaebacterium Sulfolobus solfataricus P2. Cosmids were selected from a provisional set of minimally overlapping clones, subcloned in pUC18, and sequenced using a hybrid (random plus directed) strategy to give two blocks of contiguous unique sequence, respectively, 100389 and 56105bp. These two contigs contain a total of 163 open reading frames (ORFs) in 26–29 putative operons; 56 ORFs could be identified with reasonable certainty. Clusters of ORFs potentially encode proteins of glycogen biosynthesis, oxidative decarboxylation of pyruvate, ATP‐dependent transport across membranes, isoprenoid biosynthesis, protein synthesis, and ribosomes. Putative promoters occur upstream of most ORFs. Thirty per cent of the predicted strong and medium‐strength promoters can initiate transcription at the start codon or within 10 nucleotides upstream, indicating a process of initial mRNA‐ribosome contact unlike that of most eubacterial genes. A novel termination motif is proposed to account for 15 additional terminations. The two contigs differ in densities of ORFs, insertion elements and repeated sequences; together they contain two copies of the previously reported insertion sequence ISC 1217, five additional IS elements representing four novel types, four classes of long non‐IS repeated sequences, and numerous short, perfect repeats.

A preliminary investigation of the order Bangiales (Bangiophycidae, Rhodophyta) based on sequences of nuclear small‐subunit ribosomal RNA genes

We investigated phylogenetic relationships among red algae of the order Bangiales by analysis of sequences of the nuclear gene encoding cytosolic small‐subunit ribosomal RNA in Bangia atropurpurea (Roth) C. Ag. and eight samples representing seven species of Porphyra. The ssu‐rDNA range from 1818 to 1845 nucleotides in length, with guanosine plus cytosine ratios between 47.0% and 48.6%. A group IC1 intron occurs in the B atropurpurea ssu‐rDNAs at the same position as in P. spiralis var. amplifolia Oliveira Filho et Coll and several other eukaryote ssu‐rDNAs. The nine sequences form a stable monophyletic group upon phylogenetic analysis. The ssu‐rDNA from B. atropurpurea nests stably within the Porphyra group and is closely related to P. amplissima (Kjellm.) Setchell et Hus in Hus, making the genus Porphyra paraphyletic. No correlation is seen between phylogenetic position and number of cell layers in the Porphyra thallus. We discuss possible taxonomic and evolutionary implications of these observations.

Three small, cryptic plasmids from Aeromonas salmonicida subsp. salmonicida A449.

The nucleotide sequences of three small (5.2-5.6 kb) plasmids from Aeromonas salmonicida subsp. salmonicida A449 are described. Two of the plasmids (pAsa1 and pAsa3) use a ColE2-type replication mechanism while the third (pAsa2) is a ColE1-type replicon. Insertions in the Rep protein and oriV region of the ColE2-type plasmids provide subtle differences that allow them to be maintained compatibly. All three plasmids carry genes for mobilization (mobABCD), but transfer genes are absent and are presumably provided in trans. Two of the plasmids, pAsa1 and pAsa3, carry toxin-antitoxin gene pairs, most probably to ensure plasmid stability. One open reading frame (ORF), orf1, is conserved in all three plasmids, while other ORFs are plasmid-specific. A survey of A. salmonicida strains indicates that pAsa1 and pAsa2 are present in all 12 strains investigated, while pAsa3 is present in 11 and a fourth plasmid, pAsal1, is present in 7.

Sulfolobus islandicus plasmids pRN1 and pRN2 share distant but common evolutionary ancestry

Abstract The complete sequence of the plasmid pRN2 from the thermoacidophile Sulfolobus islandicus has been determined. The plasmid was found to be circular and 6959 bp in length. S. islandicus harbors another endogenous plasmid, pRN1, and comparison of pRN1 and pRN2 revealed that these two plasmids are essentially homologous, although very distantly related. pRN1 and pRN2 share several stretches of highly conserved noncoding DNA and three common open reading frames. Two of these reading frames are likely related to replication, one encoding a large protein with a helicase domain similar to viral helicases, and the other a copy number control protein, CopG.

The Lobster Parasite Anophryoides haemophila (Scuticociliatida: Orchitophryidae): Nuclear 18S rDNA Sequence, Phylogeny and Detection Using Oligonucleotide Primers

ABSTRACT. We have determined the nucleotide sequence of the nuclear gene encoding small‐subunit ribosomal ribonucleic acid of the ciliate Anophryoides haemophila, a parasite of the American lobster Homarus americanus. The gene is 1763 bp in length, and has a guanosine‐plus‐cytosine content of 43.9%. Inferred phylogenetic frameworks strongly support the monophyly of the scuticociliates, and suggest that order Scuticociliatida should be elevated to at least subclass rank. Oligonucleotide probes based on A. haemophila ssu‐rDNA can discriminate between DNAs of A. haemophila and other investigated hymenostome ciliates, and effectively prime polymerase chain reaction‐based detection of A. haemophila deoxyribonucleic acid against at least a 1600‐fold excess of total deoxyribonucleic acid from H. americanus.

Genomic characterisation of the ichthyotoxic prymnesiophyte Chrysochromulina polylepis, and the expression of polyketide synthase genes in synchronized cultures

The widely distributed prymnesiophyte species Chrysochromulina polylepis is prominent and well known for occasional formation of ichthyotoxic blooms. The chemical structure of the C. polylepis toxin(s) has not yet been elucidated, but the associated haemolytic activity, potent membrane disruption interactions and toxicity to finfish and protists have led to the suggestion that they may be similar to the prymnesins of Prymnesium parvum. Such polyether toxins are presumably formed partially or completely via polyketide biosynthetic pathways. In this genetic study of C. polylepis, we generated and analysed a genomic DNA and a normalized cDNA library. We estimated a genome size of approximately 230 mbp based upon analysis of >1000 genomic library clones. Of the cDNA library, 3839 clones were partially sequenced and annotated, representing approximately 2900 unique contigs. We detected several genes putatively related to toxin synthesis. Thirteen putative polyketide synthase (PKS)-related gene sequences were identified and phylogenetic analysis identified two of these as containing ketoacyl domains of the modular type I PKS. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) was used to follow the expression of PKS genes over the light/dark cycle of synchronized C. polylepis cultures. This is the first study showing the expression of PKS genes in marine microalgae, in this case in the toxigenic C. polylepis.

Comparison of Next-Generation Sequencing Platforms for Clinical Testing of Non-Small Cell Lung Cancer

Personalized treatment of lung cancer using therapies that target activating oncogenic mutations such as EGFR and ALK has become the standard of care. Current molecular testing is routinely performed for single genes and increasingly in a multiplex format. However, the scarcity of sufficient biopsy material has necessitated a more high-throughput and comprehen - sive testing approach. Next Generation Sequencing (NGS) offers great promise as a highly sensitive method of detection for a variety of biopsy sources (tissue, blood, pleural effusions). However, there are multiple NGS platforms and panels with varying advantages and disadvan- tages. This pilot study compared four different library construction methods (Ion AmpliSeq, Illumina TruSeq, and Raindance Thunderbolts amplicon-based methods and Roche EZSeq se- quence capture method) and two different sequencing instruments (Ion Torrent PGM and Il- lumina MiSeq). A common set of ten tumor/normal pairs from lung adenocarcinoma patients were analysed by all platforms. Additional samples were analysed in subsets of the platforms. To assess the feasibility of sequencing circulating free DNA (cfDNA) from plasma and pleural effusions, two additional samples were analysed on two amplicon-based platforms. A bioin- formatic pipeline for automated sequence data analysis was developed using the Galaxy en- vironment. To determine the most cost-effective, technically streamlined library construction and sequencing method, we compared coverage statistics, sensitivity, variant detection, and workflow for all platforms.

Role of MicroRNAs in Progression and Recurrence of Early-Stage Lung Adenocarcinoma

Lung cancer is the leading cause of cancer-related death worldwide, and the majority of cases (77%) are not diagnosed until the disease has spread to regional lymph nodes or distant sites. Even among Non-Small Cell Lung Cancer (NSCLC) adenocarcinoma patients who have been diagnosed early and where there has been no spread to lymph nodes, recurrence after surgical intervention is high. Improved understanding of the molecular alterations involved in aggressivity and recurrence of these tumors may provide better biomarkers for the identifica - tion of patients who would benefit from adjuvant chemotherapy. By comparing the expression of microRNAs in advanced Stage II/III tumors with those expressed in earlier Stage I tumors, we aimed to identify differentially expressed molecular biomarkers that could underly progres- sion and recurrence of Stage I tumors. This pilot study utilized TaqMan qPCR assays to assess the expression of microRNAs in tumor tissue, matched normal tissue and plasma samples from Stage I and Stage II/III lung adenocarcinoma patients. Seven microRNAs were identified from plasma that could distinguish between patients with Stage I and Stage II/III adenocarcinoma. The up-regulation of miR-29a in plasma of patients with later-stage adenocarcinoma would result in enhanced expression of several molecules involved in integrin signaling, migration and proliferation. Analysis of differential expression of microRNAs in later-stage compared to early-stage lung adenocarcinoma implicates focal adhesion and ECM-receptor pathways in progression and recurrence. Plasma miR-29a is a promising biomarker that can be assessed non-invasively and whose clinical utility should be pursued.

An Unbalanced Exon-Expression qPCR-based Assay for Detection of ALK Translocation (Fusion) in Lung Cancer

1National Research Council Halifax, 1411 Oxford Street, Halifax, NS B3H 3Z1, Canada 2Department of Pathology, Queen Elizabeth II Health Science Center and Dalhousie University, 5788 University Ave., Halifax, NS B3H 1V8, Canada *Corresponding author Rama K. Singh, PhD Human Health Therapeutics Portfolio National Research Council Canada 1411 Oxford Street Halifax, Nova Scotia B3H 3Z1, Canada Tel. (902) 426-3802 Fax: (902) 426-9413 E-mail: rama.singh@nrc-cnrc.gc.ca