High Number Of Sequences Research Articles

Diversity and abundance of glycosyl hydrolase family 5 in the North Atlantic Ocean

The diversity and abundance of glycosyl hydrolase family 5 (GH5) were studied in the North Atlantic Ocean. This family was chosen because of the large number of available sequences from cultured bacteria, the variety of substrates it targets, and the high number of similar sequences in the Sargasso Sea environmental genome database. Three clone libraries of a GH5 subcluster were constructed from the Mid-Atlantic Bight and the eastern and western North Atlantic Ocean. The two North Atlantic Ocean libraries did not differ from each other but both were significantly less diverse than the Mid-Atlantic Bight library. The abundance of GH5 genes estimated by quantitative PCR was positively correlated with chlorophyll concentrations in the eastern part of a transect from Fort Pierce, Florida, to the Azores and in a depth profile, suggesting that the supply of labile organic material selects for GH5-bearing bacteria in these waters. However, the data suggest that only <1% of all bacteria harbor the GH5 subcluster. These and other data suggest that the hydrolysis of polysaccharides requires complicated multi-enzyme systems.

Mitochondrial Introgressions into the Nuclear Genome of the Domestic Cat

Translocation of mtDNA into the nuclear genome, also referred to as numt, was first reported in the domestic cat (Felis catus) by Lopez et al. (1994). The Lopez-numt consisted of a translocation of 7.9 kbp of mtDNA that inserted into the domestic cat chromosome D2 around 1.8 million years ago. More than a decade later, the release of the domestic cat whole-genome shotgun sequences (1.9x coverage) provides the resource to obtain more comprehensive insight into the extent of mtDNA transfer over time in the domestic cat genome. MegaBLAST searches revealed that the cat genome harbors a wide variety of numts (298 320 bp), one-third of which likely correspond to the Lopez-numt tandem repeat, whereas the remaining numts are probably derived from multiple independent insertions, which in some cases were followed by segmental duplication after insertion in the nucleus. Numts were detected across most cat chromosomes, but the number of numts assigned to chromosomes is underestimated due to the relatively high number of numt sequences with insufficient flanking sequence to map. The catalog of cat numts provides a valuable resource for future studies in Felidae species, including its use as a tool to avoid numt contaminations that may confound population genetics and phylogenetic studies.

A methodology for the quantitative risk assessment of major accidents triggered by seismic events

A procedure for the quantitative risk assessment of accidents triggered by seismic events in industrial facilities was developed. The starting point of the procedure was the use of available historical data to assess the expected frequencies and the severity of seismic events. Available equipment-dependant failure probability models (vulnerability or fragility curves) were used to assess the damage probability of equipment items due to a seismic event. An analytic procedure was subsequently developed to identify, evaluate the credibility and finally assess the expected consequences of all the possible scenarios that may follow the seismic events. The procedure was implemented in a GIS-based software tool in order to manage the high number of event sequences that are likely to be generated in large industrial facilities. The developed methodology requires a limited amount of additional data with respect to those used in a conventional QRA, and yields with a limited effort a preliminary quantitative assessment of the contribution of the scenarios triggered by earthquakes to the individual and societal risk indexes. The application of the methodology to several case-studies evidenced that the scenarios initiated by seismic events may have a relevant influence on industrial risk, both raising the overall expected frequency of single scenarios and causing specific severe scenarios simultaneously involving several plant units.

Retrotransposon-based S-SAP as a platform for the analysis of genetic variation and linkage in globe artichoke

A high copy number of retrotransposon sequences are present and widely dispersed in plant genomes. Their activity generates a considerable degree of sequence polymorphism. Here, we report the cloning of CYRE-5, a long-terminal repeat carrying retrotransposon-like sequence in Cynara cardunculus L., and its exploitation to develop a DNA fingerprinting assay across 22 accessions, including both cultivated (globe artichoke and cultivated cardoon) and wild (wild cardoon) types. The effectiveness of the sequence-specific amplified polymorphism (S-SAP) platform is compared with that of amplified fragment length polymorphism (AFLP). A genetic linkage analysis, based on a hybrid population between 2 globe artichoke varietal types, resulted in the inclusion of 29 S-SAP loci in the core genetic map, confirming their dispersed distribution across the globe artichoke genome.

JAFA: a protein function annotation meta-server

With the high number of sequences and structures streaming in from genomic projects, there is a need for more powerful and sophisticated annotation tools. Most problematic of the annotation efforts is predicting gene and protein function. Over the past few years there has been considerable progress in automated protein function prediction, using a diverse set of methods. Nevertheless, no single method reports all the information possible, and molecular biologists resort to ‘shopping around’ using different methods: a cumbersome and time-consuming practice. Here we present the Joined Assembly of Function Annotations, or JAFA server. JAFA queries several function prediction servers with a protein sequence and assembles the returned predictions in a legible, non-redundant format. In this manner, JAFA combines the predictions of several servers to provide a comprehensive view of what are the predicted functions of the proteins. JAFA also offers its own output, and the individual programs' predictions for further processing. JAFA is available for use from .

Unexpectedly high bacterial diversity in arctic tundra relative to boreal forest soils, revealed by serial analysis of ribosomal sequence tags.

Arctic tundra and boreal forest soils have globally relevant functions that affect atmospheric chemistry and climate, yet the bacterial composition and diversity of these soils have received little study. Serial analysis of ribosomal sequence tags (SARST) and denaturing gradient gel electrophoresis (DGGE) were used to compare composite soil samples taken from boreal and arctic biomes. This study comprises an extensive comparison of geographically distant soil bacterial communities, involving the analysis of 12,850 ribosomal sequence tags from six composite soil samples. Bacterial diversity estimates were greater for undisturbed arctic tundra soil samples than for boreal forest soil samples, with the highest diversity associated with a sample from an extreme northern location (82(o)N). The lowest diversity estimate was obtained from an arctic soil sample that was disturbed by compaction and sampled from a greater depth. Since samples from the two biomes did not form distinct clusters on the basis of SARST data and DGGE fingerprints, factors other than latitude likely influenced the phylogenetic compositions of these communities. The high number of ribosomal sequences analyzed enabled the identification of possible cosmopolitan and endemic bacterial distributions in particular soils.

Mitochondrial DNA and retroviral RNA analyses of archival oral polio vaccine (OPV CHAT) materials: evidence of macaque nuclear sequences confirms substrate identity

Inoculation of live experimental oral poliovirus vaccines (OPV CHAT) during the 1950s in central Africa has been proposed to account for the introduction of HIV into human populations. For this to have occurred, it would have been necessary for chimpanzee rather than macaque kidney epithelial cells to have been included in the preparation of early OPV materials. Theoretically, this could have led to contamination with a progenitor of HIV-1 derived from a related simian immunodeficiency virus of chimpanzees (SIV CPZ). In this article we present further detailed analyses of two samples of OPV, CHAT 10A-11 and CHAT 6039/Yugo, which were used in early human trials of poliovirus vaccination. Recovery of poliovirus by culture techniques confirmed the biological viability of the vaccines and sequence analysis of poliovirus RNA specifically identified the presence of the CHAT strain. Independent nested sets of oligonucleotide primers specific for HIV-1/SIV CPZ and HIV-2/SIV MAC/SIV SM phylogenetic lineages, respectively, indicated no evidence of HIV/SIV RNA in either vaccine preparation, at a sensitivity of 100 RNA equivalents/ml. Analysis of cellular substrate by the amplification of two distinct regions of mitochondrial DNA (D-loop control region and 12S ribosomal sequences) revealed no evidence of chimpanzee cellular sequences. However, this approach positively identified rhesus and cynomolgus macaque DNA for the CHAT 10A-11 and CHAT 6039/Yugo vaccine preparations, respectively. Analysis of multiple clones of mtDNA 12S rDNA indicated a relatively high number of nuclear mitochondrial DNA sequences (numts) in the CHAT 10A-11 material, but confirmed the macaque origin of cellular substrate used in vaccine preparation. These data re-inforce earlier findings on this topic providing no evidence to support the contention that poliovirus vaccination was responsible for the introduction of HIV into humans and sparking the AIDS pandemic.

The pathogenMycobacterium marinum, a faster growing close relative ofMycobacterium tuberculosis, has a single rRNA operon per genome

Although Mycobacterium marinum and Mycobacterium tuberculosis are very closely related they differ significantly in their growth rates. The Type strain of M. marinum and one clinical isolate were investigated and, like M. tuberculosis, were found to have a single rRNA (rrn) operon per genome located downstream from murA gene and controlled by two promoters. No sequence differences were found that account for the difference in the growth rates of the two species. We infer that M. tuberculosis has the capacity to synthesize rRNA much faster than it actually does; and propose that the high number of insertion sequences in this species attenuate growth rate to lower values.

The pathogen Mycobacterium marinum, a faster growing close relative of Mycobacterium tuberculosis, has a single rRNA operon per genome

Although Mycobacterium marinum and Mycobacterium tuberculosis are very closely related they differ significantly in their growth rates. The Type strain of M. marinum and one clinical isolate were investigated and, like M. tuberculosis, were found to have a single rRNA ( rrn) operon per genome located downstream from murA gene and controlled by two promoters. No sequence differences were found that account for the difference in the growth rates of the two species. We infer that M. tuberculosis has the capacity to synthesize rRNA much faster than it actually does; and propose that the high number of insertion sequences in this species attenuate growth rate to lower values.

Quantitative relationships for specific growth rates and macromolecular compositions of Mycobacterium tuberculosis, Streptomyces coelicolor A3(2) and Escherichia coli B/r: an integrative theoretical approach.

Further understanding of the physiological states of Mycobacterium tuberculosis and other mycobacteria was sought through comparisons with the genomic properties and macromolecular compositions of Streptomyces coelicolor A3(2), grown at 30 degrees C, and Escherichia coli B/r, grown at 37 degrees C. A frame of reference was established based on quantitative relationships observed between specific growth rates ( micro ) of cells and their macromolecular compositions. The concept of a schematic cell based on transcription/translation coupling, average genes and average proteins was developed to provide an instantaneous view of macromolecular synthesis carried out by cells growing at their maximum rate. It was inferred that the ultra-fast growth of E. coli results from its ability to increase the average number of rRNA (rrn) operons per cell through polyploidy, thereby increasing its capacity for ribosome synthesis. The maximum growth rate of E. coli was deduced to be limited by the rate of uptake and consumption of nutrients providing energy. Three characteristic properties of S. coelicolor A3(2) growing optimally ( micro =0.30 h(-1)) were identified. First, the rate of DNA replication was found to approach the rate reported for E. coli ( micro =1.73 h(-1)); secondly, all rrn operons were calculated to be fully engaged in precursor-rRNA synthesis; thirdly, compared with E. coli, protein synthesis was found to depend on higher concentrations of ribosomes and lower concentrations of aminoacyl-tRNA and EF-Tu. An equation was derived for E. coli B/r relating micro to the number of rrn operons per genome. Values of micro =0.69 h(-1) and micro =1.00 h(-1) were obtained respectively for cells with one or two rrn operons per genome. Using the author's equation relating the number of rrn operons per genome to maximum growth rate, it is expected that M. tuberculosis with one rrn operon should be capable of growing much faster than it actually does. Therefore, it is suggested that the high number of insertion sequences in this species attenuates growth rate to still lower values.

The alternation of different morphotypes in the seasonal cycle of the toxic diatom Pseudo-nitzschia galaxiae

The marine diatom Pseudo-nitzschia galaxiae Lundholm et Moestrup has been recently described from Mexican and Australian plankton. In this paper, we illustrate the considerable morphological variability of the species in the Mediterranean Sea and present first evidence of its toxicity. In addition to lanceolate cells 25–41 μm long, which fit the original description of the species, markedly larger (<82 μm) and smaller (>10 μm) specimens are commonly recorded. Cells of the largest size have almost parallel valve margins, while smaller specimens have extremely short rostrate ends and do not form colonies. Despite remarkable differences in shape and size, the typical ultrastructure of the species was observed for the different size classes in culture and in natural samples. In culture, cell length decreased at a rate of 1.1–2.1 μm per month. Liquid chromatography–mass spectrometry (LC–MS) analyses revealed the presence of domoic acid (DA) at very low levels in two of seven strains analyzed. LSU rDNA analysis confirmed the identity of the species and showed a very low genetic variability for the strains from the Gulf of Naples, with no relationships with size and overall shape of the cells. A relatively high number (53) of Pseudo-nitzschia sequences were considered in the phylogenetic analysis, yet the relationships among species remain unclear, probably in relation with a recent speciation process in the genus. In natural samples, P. galaxiae populations of different cell sizes occurred at different times over the year, with smaller cells found in winter and early spring, and medium and larger cells peaking in late spring–summer. The maximum concentration value in the Gulf of Naples was recorded in May 1985 (9.4×10 6 cells l −1). From the analysis of a high number of both natural and culture samples, it is concluded that size and shape variations are indicative of different stages of the life cycle of P. galaxiae, which exhibit a synchronized and seasonal occurrence at the interannual scale.

Proteome-wide classification and identification of mammalian-type GPCRs by binary topology pattern

G protein-coupled receptors (GPCRs), a large eukaryotic protein family, have proved difficult to comprehensively detect and functionally identify by homology searches and domain detection, because they are highly divergent and their sequences share strikingly little similarity. Transmembrane (TM) topology pattern analysis has been used to classify TM proteins, and such patterns are conserved within GPCRs of similar function. Here, we developed a stepwise binary topology pattern (BTP) method for GPCR classification and identification and used it to identify and classify mammalian-type GPCRs in the genomes of 10 different eukaryotic species. A binary topology pattern was obtained for each functional class or group by assigning binary loop threshold lengths of ‘0’ (short loop) or ‘1’ (long loop). The GPCR-classification ability of the BTP method had quite high accuracies for classifying GPCR functions at the class level (Classes A, B, C, Frizzled/Smoothened, Non-GPCR, based on the GPCRDB classification scheme), with many classes being classified with 100% accuracy. Sufficiently high accuracies were also maintained at the functional group level, 0.945 over 15 functional groups. Proteome-wide mammalian-type GPCR searches in 10 eukaryotic genomes ( H. sapiens, M. musculus, F. rubripes, C. intestinalis, A. thaliana, D. melanogaster, A. gambiae, C. elegans, P. falciparum, S. cerevisiae) using the BTP method showed much higher classification/identification in non-mammalian genomes than typical BLAST searches, in which a higher number of sequences were classified as Non-GPCR. This stepwise BTP method should prove useful for the identification and functional classification of GPCRs from the genomes of a wide range of species.

Microbial Communities in Composite Biofilms Participating in the Degradation of PCB

A moorland soil site polluted with PCB showed a high diversity ofmetabolically active bacteria. Beside frequent types of 16S rRNAsequences similar to those of the species ofSphingomonasand the Acidobacterium phylum an unusual high number ofsequences from the genus Burkholderia were found. Burkholderia was also the main genus in isolates enriched onbiphenyl or various chlorobenzoates. In microcosm experimentssterilized surfaces exposed to PCB polluted soil always showed thepresence of clay aggregates formed by bacteria attached to thesubstratum. The bacteria use the PCB loaded clay colloids astransport medium for the water insoluble substrate to get accessto the carbon source. This is a novel mechanism of how bacteria dealwith hydrophobic substrates.

Efficient transduction and long-term retroviral expression of the melanoma-associated tumor antigen tyrosinase in CD34(+) cord blood-derived dendritic cells.

Differentiation of genetically modified CD34(+) hematopoietic stem cells into dendritic cells (DCs) will contribute to the development of immunotherapeutic anticancer protocols. Retroviral vectors that have been used for the transduction of CD34(+) cells face the problem of gene silencing when integrated into the genome of repopulating stem cells. We reasoned that a high copy number of retroviral DNA sequences might overcome silencing of transgene expression during expansion and differentiation of progenitor cells into functional DCs. To prove this, we utilized a retroviral vector with bicistronic expression of the melanoma-associated antigen tyrosinase and the enhanced green fluorescent protein (EGFP). Human cord blood CD34(+) cells were transduced with vesicular stomatitis virus G-protein (VSV-G) pseudotyped Moloney murine leukemia virus (MoMuLV) particles using 100-150 multiplicity of infection. During expansion of transduced cells with immature phenotype, transgene expression was strongly silenced, but upon differentiation into mature DCs, residual transgene expression was retained. Intracellular processing of the provirally expressed tyrosinase was tested in a chromium release assay utilizing a cytotoxic T cell clone specific for a HLA-A*0201-restricted tyrosinase peptide. We suggest that retroviral transduction of tumor-associated antigens in hematopoietic progenitor cells and subsequent differentiation into DCs is a suitable basis for the development of potent anti-tumor vaccines.

Improving quality of expressed sequence tag (EST) databases: recovery of reversed, antisense cDNA sequences.

Expressed sequence tag (EST) databases contain a significant number (5-20%) of reversed, antisense, cDNA sequences that can be recognized by the label "reversed clone: similarity on wrong strand" in the annotations to the sequence. Despite this high number of altered sequences, no attempt has been made to explain the alteration in molecular terms, or to evaluate their effect on the quality of the information curated in EST databases. In this paper we try to explain the way these altered sequences are originated, and propose a plausible mechanism: a "double priming" of the first strand oligo-dT primer at both ends of nascent cDNAs. In this way, a symmetrical cDNA intermediate is generated, an intermediate that can be cloned after partial digestion with the restriction enzyme used for the directional cloning. Furthermore, when "secondary" priming takes place inside the cDNA, the chain synthesized is prone to be truncated prematurely, with the subsequent loss of upstream information. One of the most subtle effects of this cloning alteration is the generation of virtual open reading frames (ORFs) in sequences with no homologues available for comparison. Nevertheless, and according to our model and our data, the "double priming mechanism" does not shift the ORF effected, so antisense sequences should be considered as normal ones after a simple transformation in their inverse-complementary forms.

Structure of adeno-associated virus vector DNA following transduction of the skeletal muscle.

The skeletal muscle provides a very permissive physiological environment for adeno-associated virus (AAV) type 2-mediated gene transfer. We have studied the early steps leading to the establishment of permanent transgene expression, after injection of recombinant AAV (rAAV) particles in the quadriceps muscle of mice. The animals received an rAAV encoding a secreted protein, murine erythropoietin (mEpo), under the control of the human cytomegalovirus major immediate-early promoter and were sacrificed between 1 and 60 days after injection. The measurement of plasma Epo levels and of hematocrits indicated a progressive increase of transgene expression over the first 2 weeks, followed by a stabilization at maximal plateau values. The rAAV sequences were analyzed by Southern blotting following neutral or alkaline gel electrophoresis of total DNA from injected muscles. While a high number of rAAV sequences were detected during the first 5 days following the injection, only a few percent of these sequences was retained in the animals analyzed after 2 weeks, in which transgene expression was maximal. Double-stranded DNA molecules resulting from de novo second-strand synthesis were detected as early as day 1, indicating that this crucial step of AAV-mediated gene transfer is readily accomplished in the muscle. The templates driving stable gene expression at later time points are low in copy number and structured as high-molecular-weight concatemers or interlocked circles. The presence of the circular form of the rAAV genomes at early time points suggests that the molecular transformations involved in the formation of stable concatemers may involve a rolling-circle type of DNA replication.

Degree and distribution of variability in the 5' untranslated, E1, E2/NS1 and NS5 regions of the hepatitis C virus (HCV).

Hepatitis C virus (HCV) shows a high degree of variability resulting in many different variants. In this work we described the variability of several subgenomic fragments from the 5' untranslated region (5'-UTR) and E1, E2/NS1 and NS5 regions comparing, for every position, all the sequences published in GenBank v. 88 (July 1995) as well as new sequences obtained in this work. Variability was determined in two ways. First, we analysed the degree and type of substitutions found in these regions. Second, we defined the most variable and conserved segments in each region and compared our prediction with previous studies. Our results confirm that HCV variability changes along the different regions. Although we found four variable domains in the 5'-UTR, this region was the only one to contain conserved domains. Envelope (E1, E2/NS1) and NS5 regions showed high variability throughout; however, we were able to define six and three hypervariable domains, respectively. The degree and distribution of variability established in this work is supported by the high number of sequences and the different types included in the study. Knowledge of how variability is distributed along the different regions of the HCV genome could be of use in the design of new diagnostic and therapeutic strategies against HCV infection.

Interviewer Effects in the Measurement of Personal Network Size

Methods for delineating personal networks in surveys contain complex instructions for the interviewers. It is assumed that the interviewers' experience and education influence their ability to follow these instructions. The magnitude of the interviewer effects on the personal network size has been investigated, and differences among interviewers have been explained on the basis of their experience and education. The data are from a survey among 4,059 older adults in the Netherlands interviewed in 1992 by 87 interviewers. A strong interviewer effect was observed. Furthermore, the results of a multilevel regression analysis showed that, controlled for respondent characteristics, well-educated interviewers with minor experience prior to the project and major experience within the project (i.e., the high sequence number of the interview) generated relatively large networks.

A repetitive DNA sequence common to the different B chromosomes of the genus Brachycome

Dot-like micro B chromosomes of Brachycome dichromosomatica were analysed for their sequence composition. Southern hybridization patterns of a total micro B probe to genomic DNA from plants with and without micro Bs demonstrated that the micro Bs shared sequences with the A chromosomes. In addition to telomere, rDNA and common A and B chromosome sequences, a new B-specific, highly methylated tandem repeat (Bdm29) was detected. After in situ hybridization with Bdm29 the entire micro B chromosome was labelled and clustering of the condensed micro Bs could be observed at interphase. A high number of Bdm29-like sequences were also found in the larger B chromosomes of B. dichromosomatica and in other Bs within the genus Brachycome.

A tale of two islands: population history and mitochondrial DNA sequence variation of Bioko and São Tomé, Gulf of Guinea.

The hypervariable segment I of the control region of the mtDNA was sequenced in 45 unrelated individuals from Bioko and 50 from São Tomé, two islands in the Gulf of Guinea that have had very different settlement patterns: Bioko was colonized around 10000 BP, while São Tomé was first settled by the Portuguese, who brought African slaves to the island. Two different patterns of sequence variation are evident and are also clearly a consequence of their very different demographic histories. The Bubi present a low genetic diversity and it is likely that the island was colonized by a small number of individuals with small later migration. São Tomeans might be considered a subset of a mainland African population relocated to the island. They present high genetic diversity with a high number of sequences being shared with many continental populations. This study, with knowledge of the population history in island populations, strengthens the genetic approach to unravel past demographic events.