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Rhodococcus folensis sp. nov., an orange-red-pigmented bacterium from mining soil.

Mining-impacted environments represent chemically complex ecosystems that may harbor metabolically versatile and pigment-producing microorganisms. During a survey of pigment-producing bacteria from abandoned mining soil in Trabzon, Türkiye, a red-pigmented strain, designated FMA22T, was isolated and characterized using a polyphasic taxonomic approach. 16 S rRNA gene sequence analysis placed the strain within the genus Rhodococcus, showing the highest similarity to R. corynebacterioides DSM 20,151T (99.57%), R. kroppenstedtii DSM 44908ᵀ (99.06%) and R. trifolii T8T (98.96%). The strain was Gram-stain-positive, aerobic and non-motile, and grew at 4-40 °C. Polar lipids included phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, phosphatidylcholine, five unidentified glycolipids, four unidentified lipids, one unidentified phospholipid and one unidentified phosphoglycolipid; MK-8(H2) was the major respiratory quinone. Major fatty acids were C18:1 ω9c, summed feature 3 (C16:1 ω7c/C16:1 ω6c) and C16:0. ANI and dDDH values with the closest relatives were below 76.8% and 20.5%, respectively. The draft genome (4.23 Mb; 67.2 mol% G + C; 4,106 CDSs) harbors a terpene-associated carotenoid cluster containing crtB, crtI and crtY. The orange-red pigment (λmax = 475 nm) showed antioxidant activity (DPPH SC₅₀ = 5.38 mg mL⁻¹; FRAP = 4.34 µmol TE g⁻¹) and weak but measurable HIV-1 reverse transcriptase inhibition (IC₅₀ = 22 mg mL⁻¹). These data support the proposal of Rhodococcus folensis sp. nov., with FMA22ᵀ (= LMG 34144ᵀ = DSM 120048ᵀ) as the type strain.

Soil Microbiology↗

A comprehensive radiation hybrid map of bovine Chromosome 26 (BTA26): comparative chromosomal organization between HSA10q and BTA26 and BTA28.

In this study, we present a comprehensive 3000-rad radiation hybrid (RH) map of bovine Chromosome (Chr) 26 (BTA26) with 80 markers including 50 genes or ESTs: 44 have an ortholog mapping to human Chr 10 (HSA10) and 29 to mouse Chr (MMU) 7, 10, and 19. Moreover, 12 other HSA10 genes were integrated in a newly developed RH map of BTA28 (seven represent new assignments). The available draft of the mouse genome allowed us to present a detailed picture of the distribution of conserved synteny segments among the three species (human, cattle, and mouse) and to propose a simple model of the comparative chromosomal organization between the long arm of HSA10 and BTA26 and 28. Finally, the INRA bovine BAC library was screened for most of the BTA26 markers considered in this study to provide anchors for the bovine physical map.

Animals↗

Association studies between microsatellite markers within the gene encoding human 11beta-hydroxysteroid dehydrogenase type 1 and body mass index, waist to hip ratio, and glucocorticoid metabolism.

Two isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) interconvert active cortisol (F) and inactive cortisone (E). 11beta-HSD1 is an oxo-reductase (E to F) expressed in several glucocorticoid target tissues, including liver and adipose tissue, where it facilitates glucocorticoid-induced gluconeogenesis and adipocyte differentiation, respectively. We have isolated a full-length HSD11B1 genomic clone; the gene is more than 30 kb in length, not 9 kb in length as previously reported, principally due to a large intron 4. Two polymorphic (CA)(n) repeats have been characterized within intron 4: a CA(19) repeat 2.7 kb 3' of exon 4 and a CA(15) repeat 3 kb 5' of exon 5. The microsatellites, CA(19) and CA(15), were PCR amplified using fluorescent primers and were genotyped on an ABI 377 DNA sequencer from DNA of 413 normal individuals enrolled in the MONICA study of cardiovascular risk factors and 557 Danish men (ADIGEN study), of whom 234 were obese [body mass index (BMI), >/=31 kg/m(2) ] at draft board examination and 323 were randomly selected controls from the draftee population with BMI below 31 kg/m(2) (mean +/- SE, 21.7 +/- 0.41). Genotypic data from the normal MONICA cohort was compared with gender, 5beta-tetrahydrocortisol+5alpha-tetrahydrocortisol/tetrahydrocortisone ratio, and waist to hip (W:H) ratio. When analyzed by allele length (0, 1, or 2 short alleles) for the CA(19) marker, there was a trend toward a higher 5beta-tetrahydrocortisol+5alpha-tetrahydrocortisol/tetrahydrocortisone ratio (P = 0.058) and an increased W:H ratio (2 vs. 0.1 short; P(c) = 0.10) with overrepresentation of short alleles. The opposite was true for the CA(15) locus, with longer alleles at this locus predicting increased 11beta-HSD1 activity, particularly in females. Genotypic data from the ADIGEN case-control population was compared with clinical markers of obesity such as BMI and W:H ratio. There was no significant difference in the distribution of either microsatellite marker between lean and obese groups. Allele distributions were binomial, as seen for the MONICA cohort, and the data were split accordingly (zero, one, or two short alleles). No significant association was seen between grouped alleles and the clinical parameters. No association was observed between HSD11B1 genotype and BMI in either population. These data suggest that 11beta-HSD1 is not a major factor in explaining genetic susceptibility to obesity per se. However, weak associations between HSD11B1 genotype, increased 11beta-HSD1 activity, and W:H ratio suggest that polymorphic variability at the HSD11B1 locus may influence susceptibility to central obesity through enhanced 11beta-HSD1 activity (E to F conversion) in visceral adipose tissue.

11-beta-Hydroxysteroid Dehydrogenase Type 2↗

Two types of FtsZ proteins in mitochondria and red-lineage chloroplasts: the duplication of FtsZ is implicated in endosymbiosis.

The ancestors of plastids and mitochondria were once free-living bacteria that became organelles as a result of endosymbiosis. According to this theory, a key bacterial division protein, FtsZ, plays a role in plastid division in algae and plants as well as in mitochondrial division in lower eukaryotes. Recent studies have shown that organelle division is a process that combines features derived from the bacterial division system with features contributed by host eukaryotic cells. Two nonredundant versions of FtsZ, FtsZ1 and FtsZ2, have been identified in green-lineage plastids, whereas most bacteria have a single ftsZ gene. To examine whether there is also more than one type of FtsZ in red-lineage chloroplasts (red algal chloroplasts and chloroplasts that originated from the secondary endosymbiosis of red algae) and in mitochondria, we obtained FtsZ sequences from the complete sequence of the primitive red alga Cyanidioschyzon merolae and the draft sequence of the stramenopile (heterokont) Thalassiosira pseudonana. Phylogenetic analyses that included known FtsZ proteins identified two types of chloroplast FtsZ in red algae (FtsZA and FtsZB) and stramenopiles (FtsZA and FtsZC). These analyses also showed that FtsZB emerged after the red and green lineages diverged, while FtsZC arose by the duplication of an ftsZA gene that in turn descended from a red alga engulfed by the ancestor of stramenopiles. A comparison of the predicted proteins showed that like bacterial FtsZ and green-lineage FtsZ2, FtsZA has a short conserved C-termmal sequence (the C-terminal core domain), whereas FtsZB and FtsZC, like the green-lineage FtsZ1, lack this sequence. In addition, the Cyanidioschyzon and Dictyostelium genomes encode two types of mitochondrial FtsZ proteins, one of which lacks the C-terminal variable domain. These results suggest that the acquisition of an additional FtsZ protein with a modified C terminus was common to the primary and secondary endosymbioses that produced plastids and that this also occurred during the establishment of mitochondria, presumably to regulate the multiplication of these organelles.

Algal Proteins↗

Identification of open reading frames unique to a select agent: Ralstonia solanacearum race 3 biovar 2.

An 8x draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10(-13)) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.

Arginine↗

Allelic imbalance on chromosome 10 in rat endometrial adenocarcinomas.

Earlier work using comparative genome hybridization (CGH) has shown that rat chromosome 10 (RNO10) is frequently involved in cytogenetic aberrations in BDII rat endometrial adenocarcinomas (EAC). Relative reduction in copy number (chromosomal deletions) was seen in the proximal to middle part of the chromosome, whereas there were increases in copy number in the distal part. The occurrence of RNO10 aberrations was further analyzed in DNA from primary tumor material from 42 EACs and 3 benign endometrial tumors using allelotyping of microsatellite markers. We found frequently that there were 4 quite distinct RNO10 regions that exhibited allelic imbalance. Based on these findings we believe that genes with relevance to EAC tumor development are situated in each of these chromosome regions. Extrapolation of our microsatellite marker data to the rat draft DNA sequence will facilitate the definition of the regions at the level of the DNA and to select and characterize candidate genes within each of the affected chromosome regions.

Adenocarcinoma↗

Has the yo-yo stopped? An assessment of human protein-coding gene number.

Since the identification of approximately 25,000 proteins from the draft human genome assembly in 2001, estimates of the total have oscillated between 30,000 and 70,000. The recently announced genome closure has not generated a consensus gene count despite this being a key parameter for many areas of biology including drug target discovery and characterization of the human proteome. Contrary to earlier predictions of constitutive under-detection for eukaryotic genes, the latest model organism updates have produced minor increases in the worm but fly and yeast gene numbers have decreased. The postdraft, precompletion interval has produced large increases in human transcript coverage, continuous improvements in genome assembly and refinements in automated genomic annotation. Notably these enhancements have resulted in an Ensembl human protein-coding gene number of 22,184, a decrease of 1862 since the first release. Longitudinal database surveys indicate that redundancy-reduced human mRNA and protein collections are flattening out at approximately 28,000, although Ensembl maps approximately 20,000 known sequences. Observations suggest high-throughput cloning projects are predominantly extending known genes or sampling new splice forms and novel protein discovery has slowed to a trickle. The hypothesis that substantial numbers of short proteins remain experimentally and computationally undetected in mammalian genomes is neither supported by sequence data nor by the extensive homology between mouse and human proteins. Aggregating the independent annotations for complete transcripts from seven completed human chromosomes extrapolates to approximately 25,000 genes. The inclusion of partial putative genes would increase this to above 30,000 but recent data suggest these represent predominantly nonprotein-coding transcripts. Mass spectrometry-based proteomics has already verified more than 10% of human genes but has not identified significant numbers of unpredicted proteins. The available data are thus converging to a basal protein-coding gene number well below 30,000, which could even be as low as 25,000.

Chromosomes, Human↗

An active DNA transposon family in rice.

The publication of draft sequences for the two subspecies of Oryza sativa (rice), japonica (cv. Nipponbare) and indica (cv. 93-11), provides a unique opportunity to study the dynamics of transposable elements in this important crop plant. Here we report the use of these sequences in a computational approach to identify the first active DNA transposons from rice and the first active miniature inverted-repeat transposable element (MITE) from any organism. A sequence classified as a Tourist-like MITE of 430 base pairs, called miniature Ping (mPing), was present in about 70 copies in Nipponbare and in about 14 copies in 93-11. These mPing elements, which are all nearly identical, transpose actively in an indica cell-culture line. Database searches identified a family of related transposase-encoding elements (called Pong), which also transpose actively in the same cells. Virtually all new insertions of mPing and Pong elements were into low-copy regions of the rice genome. Since the domestication of rice mPing MITEs have been amplified preferentially in cultivars adapted to environmental extremes-a situation that is reminiscent of the genomic shock theory for transposon activation.

Amino Acid Sequence↗

Functional genomics and gene microarrays--the use in research and clinical medicine.

In the year 2000, the Human Genome Project Consortium presented the first complete draft of the human genome together with Celera Genetics. Since then, the so-called "post-genome era" has started. Microarrays are capable of profiling gene expression patterns of tens of thousands of genes in a single experiment and thus allow a systematic analysis of DNA and RNA variation. They seem likely to become a standard tool of both molecular biology research and clinical diagnostics. These prospects have attracted great interest and investment from both the public and private sectors. This review introduces the principle of microarray technology and gives an overview of its current and future potential in clinical medicine.

Animals↗

New generation pharmacogenomic tools: a SNP linkage disequilibrium Map, validated SNP assay resource, and high-throughput instrumentation system for large-scale genetic studies.

Since public and private efforts announced the first draft of the human genome last year, researchers have reported great numbers of single nucleotide polymorphisms (SNPs). We believe that the availability of well-mapped, quality SNP markers constitutes the gateway to a revolution in genetics and personalized medicine that will lead to better diagnosis and treatment of common complex disorders. A new generation of tools and public SNP resources for pharmacogenomic and genetic studies--specifically for candidate-gene, candidate-region, and whole-genome association studies--will form part of the new scientific landscape. This will only be possible through the greater accessibility of SNP resources and superior high-throughput instrumentation-assay systems that enable affordable, highly productive large-scale genetic studies. We are contributing to this effort by developing a high-quality linkage disequilibrium SNP marker map and an accompanying set of ready-to-use, validated SNP assays across every gene in the human genome. This effort incorporates both the public sequence and SNP data sources, and Celera Genomics' human genome assembly and enormous resource ofphysically mapped SNPs (approximately 4,000,000 unique records). This article discusses our approach and methodology for designing the map, choosing quality SNPs, designing and validating these assays, and obtaining population frequency ofthe polymorphisms. We also discuss an advanced, high-performance SNP assay chemisty--a new generation of the TaqMan probe-based, 5' nuclease assay-and high-throughput instrumentation-software system for large-scale genotyping. We provide the new SNP map and validation information, validated SNP assays and reagents, and instrumentation systems as a novel resource for genetic discoveries.

Alleles↗

High-resolution BAC-based map of the central portion of mouse chromosome 5.

The current strategy for sequencing the mouse genome involves the combination of a whole-genome shotgun approach with clone-based sequencing. High-resolution physical maps will provide a foundation for assembling contiguous segments of sequence. We have established a bacterial artificial chromosome (BAC)-based map of a 5-Mb region on mouse Chromosome 5, encompassing three gene families: receptor tyrosine kinases (PdgfraKit-Kdr), nonreceptor protein-tyrosine type kinases (Tec-Txk), and type-A receptors for the neurotransmitter GABA (Gabra2, Gabrb1, Gabrg1, and Gabra4). The construction of a BAC contig was initiated by hybridization screening the C57BL/6J (RPCI-23) BAC library, using known genes and sequence tagged sites (STSs). Additional overlapping clones were identified by searching the database of available restriction fingerprints for the RPCI-23 and RPCI-24 libraries. This effort resulted in the selection of >600 BAC clones, 251 kb of BAC-end sequences, and the placement of 40 known and/or predicted genes within this 5-Mb region. We use this high-resolution map to illustrate the integration of the BAC fingerprint map with a radiation-hybrid map via assembled expressed sequence tags (ESTs). From annotation of three representative BAC clones we demonstrate that up to 98% of the draft sequence for each contig could be ordered and oriented using known genes, BAC ends, consensus sequences for transcript assemblies, and comparisons with orthologous human sequence. For functional studies, annotation of sequence fragments as they are assembled into 50-200-kb stretches will be remarkably valuable.

Animals↗

Construction of a cDNA microarray derived from the ascidian Ciona intestinalis.

A cDNA microarray was constructed from a basal chordate, the ascidian Ciona intestinalis. The draft genome of Ciona has been read and inferred to contain approximately 16,000 protein-coding genes, and cDNAs for transcripts of 13,464 genes have been characterized and compiled as the "Ciona intestinalis Gene Collection Release I". In the present study, we constructed a cDNA microarray of these 13,464 Ciona genes. A preliminary experiment with Cy3- and Cy5-labeled probes showed extensive differential gene expression between fertilized eggs and larvae. In addition, there was a good correlation between results obtained by the present microarray analysis and those from previous EST analyses. This first microarray of a large collection of Ciona intestinalis cDNA clones should facilitate the analysis of global gene expression and gene networks during the embryogenesis of basal chordates.

Animals↗

Structure and regulation of human troponin genes.

The recent completion of a first draft of the human genome has allowed "in silico" genome browsing to become routine. Such computer-based research is now a useful adjunct to experiments based at the bench, and is accelerating gene discovery and the analysis and understanding of genes in their genomic contexts. This review summarises recent findings on genes encoding proteins of the troponin complex. We describe the organization of the three pairs of genes which encode isoforms of troponins I and T, and discuss how this relates to their evolution and regulation. Detailed analysis of the chromosomal context of the cardiac troponin I and slow skeletal troponin T genes reveals a region of densely packed differentially expressed genes, including new genes identified by automatic genome annotation. This information is discussed within the context of detailed analysis of the best-studied gene in this region, cardiac troponin I. In this way, we illustrate the uses to which a combination of conventional bench experiments and "in silico" analyses may be put in understanding the relationship between structure and function within the genome.

Animals↗

Protein profiling of human pancreatic islets by two-dimensional gel electrophoresis and mass spectrometry.

Completion of the human genome sequence has provided scientists with powerful resources with which to explore the molecular events associated with disease states such as diabetes. Understanding the relative levels of expression of gene products, especially of proteins, and their post-translational modifications will be critical. However, though the pancreatic islets play a key role in glucose homeostasis, global protein expression data in human are decidedly lacking. We here report the two-dimensional protein map and database of human pancreatic islets. A high level of reproducibility was obtained among the gels and a total of 744 protein spots were detected. We have successfully identified 130 spots corresponding to 66 different protein entries and generated a reference map of human islets. The functionally characterized proteins include enzymes, chaperones, cellular structural proteins, cellular defense proteins, signaling molecules, and transport proteins. A number of proteins identified in this study (e.g., annexin A2, elongation factor 1-alpha 2, histone H2B.a/g/k, heat shock protein 90 beta, heat shock 27 kDa protein, cyclophilin B, peroxiredoxin 4, cytokeratins 7, 18, and 19) have not been previously described in the database of mouse pancreatic islets. In addition, altered expression of several proteins, like GRP78, GRP94, PDI, calreticulin, annexin, cytokeratins, profilin, heat shock proteins, and ORP150 have been associated with the development of diabetes. The data presented in this study provides a first-draft reference map of the human islet proteome, that will pave the way for further proteome analysis of pancreatic islets in both healthy and diabetic individuals, generating insights into the pathophysiology of this condition.

Adult↗

WormBase: new content and better access.

WormBase (http://wormbase.org), a model organism database for Caenorhabditis elegans and other related nematodes, continues to evolve and expand. Over the past year WormBase has added new data on C.elegans, including data on classical genetics, cell biology and functional genomics; expanded the annotation of closely related nematodes with a new genome browser for Caenorhabditis remanei; and deployed new hardware for stronger performance. Several existing datasets including phenotype descriptions and RNAi experiments have seen a large increase in new content. New datasets such as the C.remanei draft assembly and annotations, the Vancouver Fosmid library and TEC-RED 5' end sites are now available as well. Access to and searching WormBase has become more dependable and flexible via multiple mirror sites and indexing through Google.

Animals↗

Perspectives on the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for short tandem repeat genotyping in the post-genome era.

The recent completion of the first rough draft of the human genome has provided fundamental information regarding our genetic make-up; however, the post-genome era will certainly require a host of new technologies to address complex biological questions. In particular, a rapid and accurate approach to characterize genetic markers, including short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) is demanded. STRs are the most informative of the two polymorphisms owing to their remarkable variability and even dispersity throughout eukaryotic genomes. Mass spectrometry is rapidly becoming a significant method in DNA analysis and has high probability of revolutionizing the way in which scientists probe the human genome. It is our responsibility as biomolecular mass spectrometrists to understand the issues in genetic analysis and the capabilities of mass spectrometry so that we may fulfill our role in developing a rapid, reliable technology to answer specific biological questions. This perspective is intended to familiarize the mass spectrometry community with modern genomics and to report on the current state of mass spectrometry, specifically electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry, for characterization of STRs.

Alleles↗

An object model and database for functional genomics.

MOTIVATION: Large-scale functional genomics analysis is now feasible and presents significant challenges in data analysis, storage and querying. Data standards are required to enable the development of public data repositories and to improve data sharing. There is an established data format for microarrays (microarray gene expression markup language, MAGE-ML) and a draft standard for proteomics (PEDRo). We believe that all types of functional genomics experiments should be annotated in a consistent manner, and we hope to open up new ways of comparing multiple datasets used in functional genomics. RESULTS: We have created a functional genomics experiment object model (FGE-OM), developed from the microarray model, MAGE-OM and two models for proteomics, PEDRo and our own model (Gla-PSI-Glasgow Proposal for the Proteomics Standards Initiative). FGE-OM comprises three namespaces representing (i) the parts of the model common to all functional genomics experiments; (ii) microarray-specific components; and (iii) proteomics-specific components. We believe that FGE-OM should initiate discussion about the contents and structure of the next version of MAGE and the future of proteomics standards. A prototype database called RNA And Protein Abundance Database (RAPAD), based on FGE-OM, has been implemented and populated with data from microbial pathogenesis. AVAILABILITY: FGE-OM and the RAPAD schema are available from http://www.gusdb.org/fge.html, along with a set of more detailed diagrams. RAPAD can be accessed by registration at the site.

Abstracting and Indexing↗

The genome of the early chordate Ciona intestinalis encodes only five cytoplasmic intermediate filament proteins including a single type I and type II keratin and a unique IF-annexin fusion protein.

We screened the recently established draft genome of the early chordate Ciona intestinalis for genes encoding cytoplasmic intermediate filament (IF) proteins. The draft of the tunicate/urochordate genome contains only the five genes (IF-A, IF-B, IF-C, IF-D and IF-F) previously established by cDNA cloning. Three of these IF proteins (IF-D, IF-C, IF-A) were shown to be orthologs of vertebrate IF subfamilies I to III while two proteins (IF-B, IF-F) seemed tunicate specific. This is now firmly established for protein IF-F since the genomic data show that it arises as a fusion protein with a C-terminal annexin domain, a feature not found before in the very large collection of metazoan IF proteins. The results also confirm the previous proposal that urochordates lack orthologs of vertebrate type IV IF proteins. We discuss the striking increase of IF complexity from 5 tunicate to 65 human genes during chordate evolution. Thus the tunicate has a single keratin pair, which is expressed in the epidermis, while the human genome has at least 25 genes each for keratins I and keratins II. Finally there are four normal Ciona annexin genes in addition to the gene encoding the IF-annexin fusion proteins (IF-F).

Amino Acid Sequence↗