High incidence of cryptic repeated elements in microsatellite flanking regions of galatheid genomes and its evolutionary implications

During the development of PCR primer sets for icrosatellite marker loci from enriched genomic libraries for three squat lobster species from Galatheidae (Decapoda: Anomura); Munida rugosa (Fabricius, 1775), M. sarsi (Huus, 1935), and Galathea strigosa (Linnaeus, 1761) (collectively known as squat lobsters), a number of unforeseen problems were encountered. These included PCR amplification failure, lack of amplification consistency, and the amplification of multiple fragments. Careful examination of microsatellite containing sequences revealed the existence of cryptic repeated elements on presumed unique flanking regions. BLAST analysis of these and other VNTR containing sequences (N 5 252) indicates that these cryptic elements can be grouped into families based upon sequence similarities. The unique features characterising these families suggest that different molecular mechanisms are involved. Of particular relevance is the association of microsatellites with mobile elements. This is the first reported observation of this phenomenon in crustaceans, and it also helps to explain why microsatellite primer development in galatheids has been relatively unsuccessful to date. We suggest a number of steps that can be used to identify similar problems in microsatellite marker development for other species, and also alternative approaches for both marker development and for the study of molecular evolution of species characterised by complex genome organisation. More specifically, we argue that new generation sequencing methodologies, which capitalise on parallel and multiplexed sequencing may pave the way forward for future crustacean research.

Genomes of "phiKMV-like viruses" of Pseudomonas aeruginosa contain localized single-strand interruptions

The "phiKMV-like viruses" comprise an important genus of T7 related phages infecting Pseudomonas aeruginosa. The genomes of these bacteriophages have localized single-strand interruptions (nicks), a distinguishing genomic trait previously thought to be unique for T5 related coliphages. Analysis of this feature in the newly sequenced phage fkF77 shows all four nicks to be localized on the non-coding DNA strand. They are present with high frequencies within the phage population and are introduced into the phage DNA at late stages of the lytic cycle. The general consensus sequence in the nicks (5'-CGACxxxxxCCTAoh pCTCCGG-3') was shown to be common among all phiKMV-related phages.

Comparative analysis of Klebsiella pneumoniae genomes identifies a phospholipase D family protein as a novel virulence factor

BACKGROUND: Klebsiella pneumoniae strains are pathogenic to animals and humans, in which they are both a frequent cause of nosocomial infections and a re-emerging cause of severe community-acquired infections. K. pneumoniae isolates of the capsular serotype K2 are among the most virulent. In order to identify novel putative virulence factors that may account for the severity of K2 infections, the genome sequence of the K2 reference strain Kp52.145 was determined and compared to two K1 and K2 strains of low virulence and to the reference strains MGH 78578 and NTUH-K2044.

RESULTS: In addition to diverse functions related to host colonization and virulence encoded in genomic regions common to the four strains, four genomic islands specific for Kp52.145 were identified. These regions encoded genes for the synthesis of colibactin toxin, a putative cytotoxin outer membrane protein, secretion systems, nucleases and eukaryotic-like proteins. In addition, an insertion within a type VI secretion system locus included sel1 domain containing proteins and a phospholipase D family protein (PLD1). The pld1 mutant was avirulent in a pneumonia model in mouse. The pld1 mRNA was expressed in vivo and the pld1 gene was associated with K. pneumoniae isolates from severe infections. Analysis of lipid composition of a defective E. coli strain complemented with pld1 suggests an involvement of PLD1 in cardiolipin metabolism.

CONCLUSIONS: Determination of the complete genome of the K2 reference strain identified several genomic islands comprising putative elements of pathogenicity. The role of PLD1 in pathogenesis was demonstrated for the first time and suggests that lipid metabolism is a novel virulence mechanism of K. pneumoniae.

LaGomiCs - Lagomorph Genomics Consortium: An International Collaborative Effort for Sequencing the Genomes of an Entire Mammalian Order

The order Lagomorpha comprises about 90 living species, divided in 2 families: the pikas (Family Ochotonidae), and the rabbits, hares, and jackrabbits (Family Leporidae). Lagomorphs are important economically and scientifically as major human food resources, valued game species, pests of agricultural significance, model laboratory animals, and key elements in food webs. A quarter of the lagomorph species are listed as threatened. They are native to all continents except Antarctica, and occur up to 5000 m above sea level, from the equator to the Arctic, spanning a wide range of environmental conditions. The order has notable taxonomic problems presenting significant difficulties for defining a species due to broad phenotypic variation, overlap of morphological characteristics, and relatively recent speciation events. At present, only the genomes of 2 species, the European rabbit (Oryctolagus cuniculus) and American pika (Ochotona princeps) have been sequenced and assembled. Starting from a paucity of genome information, the main scientific aim of the Lagomorph Genomics Consortium (LaGomiCs), born from a cooperative initiative of the European COST Action “A Collaborative European Network on Rabbit Genome Biology—RGB-Net” and the World Lagomorph Society (WLS), is to provide an international framework for the sequencing of the genome of all extant and selected extinct lagomorphs. Sequencing the genomes of an entire order will provide a large amount of information to address biological problems not only related to lagomorphs but also to all mammals. We present current and planned sequencing programs and outline the final objective of LaGomiCs possible through broad international collaboration.

DNA in Amphibian and Reptile Venom Permits Access to Genomes Without Specimen Sacrifice

Amphibian defensive skin secretions and reptile venoms are rich sources of bioactive peptides with potential pharmacological/pharmaceutical applications. As amphibian and reptile populations are in rapid global decline, our research
group has been developing analytical methods that permit generation of robust molecular data from non-invasive skin secretion samples and venom samples. While previously we have demonstrated that parallel proteome and venom gland
transcriptome analyses can be performed on such samples, here we report the presence of DNA that facilitates the more widely-used applications of gene sequencing, such as molecular phylogenetics, in a non-invasive manner that circumvents specimen sacrifice. From this “surrogate” tissue, we acquired partial 12S and 16S rRNA gene sequences that are presented for illustration purposes. Thus from a single sample of amphibian skin secretion and reptile venom, robust and complementary proteome, transcriptome and genome data can be generated for applications in diverse scientific disciplines.

Gaining biological perspectives from schistosome genomes

Characterization of the genomic basis underlying schistosome biology is an important strategy for the development of future treatments and interventions. Genomic sequence is now available for the three major clinically relevant schistosome species, Schistosoma mansoni, S. japonicum and S. haematobium, and this information represents an invaluable resource for the future control of human schistosomiasis. The identification of a biologically important, but distinct from the host, schistosome gene product is the ultimate goal for many research groups. While the initial elucidation of the genome of an organism is critical for most biological research, continued improvement or curation of the genome construction should be an ongoing priority. In this review we will discuss prominent recent findings utilizing a systems approach to schistosome biology, as well as the increased use of interference RNA (RNAi). Both of these research strategies are aiming to place parasite genes into a more meaningful biological perspective.

The conserved Helix C region in the superfamily of interferon-a/interleukin-10-related cytokines corresponds to a high-affinity binding site for the HSP70 chaperone DnaK.

HSP70 chaperones mediate protein folding by ATP-dependent interaction with short linear peptide segments that are exposed on unfolded proteins. The mode of action of the Escherichia coli homolog DnaK is representative of all HSP70 chaperones, including the endoplasmic reticulum variant BiP/GRP78. DnaK has been shown to be effective in assisting refolding of a wide variety of prokaryotic and eukaryotic proteins, including the -helical homodimeric secretory cytokine interferon- (IFN-). We screened solid-phase peptide libraries from human and mouse IFN- to identify DnaK-binding sites. Conserved DnaK-binding sites were identified in the N-terminal half of helix B and in the C-terminal half of helix C, both of which are located at the IFN- dimer interface. Soluble peptides derived from helices B and C bound DnaK with high affinity in competition assays. No DnaK-binding sites were found in the loops connecting the -helices. The helix C DnaK-binding site appears to be conserved in most members of the superfamily of interleukin (IL)-10-related cytokines that comprises, apart from IL-10 and IFN-, a series of recently discovered small secretory proteins, including IL-19, IL-20, IL-22/IL-TIF, IL-24/MDA-7 (melanoma differentiation-associated gene), IL-26/AK155, and a number of viral IL-10 homologs. These cytokines belong to a relatively small group of homodimeric proteins with highly interdigitated interfaces that exhibit the strongly hydrophobic character of the interior core of a single-chain folded domain. We propose that binding of DnaK to helix C in the superfamily of IL-10-related cytokines may constitute the hallmark of a novel conserved regulatory mechanism in which HSP70-like chaperones assist in the formation of a hydrophobic dimeric "folding" interface.

Chloroplast microsatellites: new tools for studies in plant ecology and systematics

The nonrecombinant, uniparentally inherited nature of organelle genomes
makes them useful tools for evolutionary studies. However, in plants, detecting
useful polymorphism at the population level is often difficult because of the
low level of substitutions in the chloroplast genome, and because of the slow
substitution rates and intramolecular recombination of mtDNA. Chloroplast
microsatellites represent potentially useful markers to circumvent this problem
and, to date, studies have demonstrated high levels of intraspecific variability.
Here,we discuss the use of these markers in ecological and evolutionary
studies of plants, as well as highlighting some of the potential problems
associated with such use.

Polyploid measles virus with hexameric genome length

Particles of most virus species accurately package a single genome, but there are indications that the pleomorphic particles of parainfluenza viruses incorporate multiple genomes. We characterized a stable measles virus mutant that efficiently packages at least two genomes. The first genome is recombinant and codes for a defective attachment protein with an appended domain interfering with fusion-support function. The second has one adenosine insertion in a purine run that interrupts translation of the appended domain and restores function. In that genome, a one base deletion in a different purine run abolishes polymerase synthesis, but restores hexameric genome length, thus ensuring accurate RNA encapsidation, which is necessary for efficient replication. Thus, the two genomes are complementary. The infection kinetics of this mutant indicate that packaging of multiple genomes does not negatively affect growth. We also show that polyploid particles are produced in standard infections at no expense to infectivity. Our results illustrate how the particles of parainfluenza viruses efficiently accommodate cargoes of different volume, and suggest a mechanism by which segmented genomes may have evolved.

New perspectives on neoplasia and the RNA world

Key tenets of modern biology are the central place of protein in cell regulation and the flow of genetic information from DNA to RNA to protein. However, it is becoming increasingly apparent that genomes are much more complex than hitherto thought with remarkably complex regulatory systems. The notion that the fraction of the genome involved in coding protein is all that matters is increasingly being questioned as the roles of non-coding RNA (ncRNA) in cellular systems becomes recognised. The RNA world, including microRNA (miRNA), small inhibitory RNA (siRNA) and other RNA species, are now recognised as being crucial for the regulation of chromatin structure, gene expression, mRNA processing and splicing, mRNA stability and translational control. Furthermore such ncRNA systems may be perturbed in disease states and most notably in neoplasia, including in haematological malignancies. Here the burgeoning evidence for a role of miRNA in neoplasia is reviewed and the importance of understanding the RNA world emphasised. Copyright (c) 2005 John Wiley & Sons, Ltd.

Measles virus minigenomes encoding two autofluorescent proteins reveal cell-to-cell variation in reporter expression dependent on viral sequences between the transcription units.

Transcription from morbillivirus genomes commences at a single promoter in the 3' non-coding terminus, with the six genes being transcribed sequentially. The 3' and 5' untranslated regions (UTRs) of the genes (mRNA sense), together with the intergenic trinucleotide spacer, comprise the non-coding sequences (NCS) of the virus and contain the conserved gene end and gene start signals, respectively. Bicistronic minigenomes containing transcription units (TUs) encoding autofluorescent reporter proteins separated by measles virus (MV) NCS were used to give a direct estimation of gene expression in single, living cells by assessing the relative amounts of each fluorescent protein in each cell. Initially, five minigenomes containing each of the MV NCS were generated. Assays were developed to determine the amount of each fluorescent protein in cells at both cell population and single-cell levels. This revealed significant variations in gene expression between cells expressing the same NCS-containing minigenome. The minigenome containing the M/F NCS produced significantly lower amounts of fluorescent protein from the second TU (TU2), compared with the other minigenomes. A minigenome with a truncated F 5' UTR had increased expression from TU2. This UTR is 524 nt longer than the other MV 5' UTRs. Insertions into the 5' UTR of the enhanced green fluorescent protein gene in the minigenome containing the N/P NCS showed that specific sequences, rather than just the additional length of F 5' UTR, govern this decreased expression from TU2.

Characterization of White bream virus reveals a novel genetic cluster of nidoviruses.

The order Nidovirales comprises viruses from the families Coronaviridae (genera Coronavirus and Torovirus), Roniviridae (genus Okavirus), and Arteriviridae (genus Arterivirus). In this study, we characterized White bream virus (WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5' leader sequence that is identical to the 5'-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses.

Nidovirales: evolving the largest RNA virus genome.

This review focuses on the monophyletic group of animal RNA viruses united in the order Nidovirales. The order includes the distantly related coronaviruses, toroviruses, and roniviruses, which possess the largest known RNA genomes (from 26 to 32 kb) and will therefore be called ‘large’ nidoviruses in this review. They are compared with their arterivirus cousins, which also belong to the Nidovirales despite having a much smaller genome (13–16 kb). Common and unique features that have been identified for either large or all nidoviruses are outlined. These include the nidovirus genetic plan and genome diversity, the composition of the replicase machinery and virus particles, virus-specific accessory genes, the mechanisms of RNA and protein synthesis, and the origin and evolution of nidoviruses with small and large genomes. Nidoviruses employ single-stranded, polycistronic RNA genomes of positive polarity that direct the synthesis of the subunits of the replicative complex, including the RNA-dependent RNA polymerase and helicase. Replicase gene expression is under the principal control of a ribosomal frameshifting signal and a chymotrypsin-like protease, which is assisted by one or more papain-like proteases. A nested set of subgenomic RNAs is synthesized to express the 3'-proximal ORFs that encode most conserved structural proteins and, in some large nidoviruses, also diverse accessory proteins that may promote virus adaptation to specific hosts. The replicase machinery includes a set of RNA-processing enzymes some of which are unique for either all or large nidoviruses. The acquisition of these enzymes may have improved the low fidelity of RNA replication to allow genome expansion and give rise to the ancestors of small and, subsequently, large nidoviruses.

Expression of the phosphonoalanine-degradative gene cluster from Variovorax sp Pal2 is induced by growth on phosphonoalanine and phosphonopyruvate

The phosphonopyruvate hydrolase (PalA) found in Variovorax sp., Pal2, is a novel carbon-phosphorus bond cleavage enzyme, which is expressed even in the presence of high levels of phosphate, thus permitting phosphonopyruvate to be used as the sole carbon and energy source. Analysis of the regions adjacent to the palA gene revealed the presence of the five structural genes that constitute the 2-amino-3-phosphonopropionic acid (phosphonoalanine)-degradative operon. Reverse transcriptase-PCR (RT-PCR) experiments demonstrated that all five genes in the operon are transcribed as a single mRNA and that their transcription is induced by phosphonoalanine or phosphonopyruvate. Transcriptional fusions of the regulatory region of the phosphonoalanine degradative operon with the gfp gene were constructed. Expression analysis indicated that the presence of a LysR-type regulator (encoded by the palR gene) is essential for the transcription of the structural genes of the operon. Similar gene clusters were found in the sequenced genomes of six bacterial species from the Alpha-, Beta- and Gammaproteobacteria, and analysis of metagenomic libraries revealed that sequences related to palA are widely spread in the marine environment.