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The complete Escherichia coli genome sequence is now known; it should greatly facilitate the analysis of other genomes, but a lot remains to be learnt about E. coli itself. About half the genes were previously uncharacterized, but expanding databases and improving analysis methods will help predict their functions.
The porcine erythropoietin (EPO) gene and its cDNA have been cloned and characterized. The cDNA encodes a protein of 194 amino acids. The gene structure and sequence show a high degree of homology to the corresponding human and murine gene. Steroid hormone receptor binding sites are present both in the promoter and in the 3' flanking region of the gene, which also contains an oxygen-sensing sequence. The promoter lacks classical promoter elements such as TATA and CAAT boxes. Expression analyses using a competitive RT-PCR assay showed that the kidneys contain about ten times more erythropoietin mRNA than the liver in five-week-old piglets, thus indicating that the shift from mainly hepatic to mainly renal EPO production has taken place at this age. The testes showed a higher ratio of EPO mRNA to total RNA than the liver. Spleen showed very low levels of expression, while no expression of erythropoietin mRNA was detected in brain tissue, bone marrow, lung, lymph nodes, and ovaries.
Genomic sequencing was used to localise 5-methylcytosine residues in individual DNA strands of 5S rRNA genes in tobacco. The density of methylation along the sequence was high in both strands, exceeding the average methylation density of the tobacco genome. Besides methylation of CG and CNG sequences, considerable amounts of mC were found in non-symmetrical sites. Among 69 sequenced clones obtained from leaf DNA we did not detect any non-methylated clone, and Southern blot hybridisation analysis also failed to suggest the presence of methylation-free 5S rDNA units in the tobacco genome. Differences were observed among methylation patterns of individual sequenced clones. This heterogeneity reflects either heterogeneity among individual members of 5S rRNA gene cluster or differences among individual cells. Methylation of CNG and non-symmetrical sites can be efficiently reduced by treatment with dihydroxypropyladenine, an inhibitor of S-adenosylhomocysteine hydrolase.
Genomic sequences of the HLA-DQB1*03:01:01:70, -DQB1*03:01:01:77, -DQB1*03:02:01:28, -DQB1*03:02:01:29, and -DQB1*03:03:02:19 alleles.
Sponsored by the National Science Foundation and the U.S. Department of Agriculture, a wheat genome sequencing workshop was held November 10-11, 2003, in Washington, DC. It brought together 63 scientists of diverse research interests and institutions, including 45 from the United States and 18 from a dozen foreign countries (see list of participants at http://www.ksu.edu/igrow). The objectives of the workshop were to discuss the status of wheat genomics, obtain feedback from ongoing genome sequencing projects, and develop strategies for sequencing the wheat genome. The purpose of this report is to convey the information discussed at the workshop and provide the basis for an ongoing dialogue, bringing forth comments and suggestions from the genetics community.
INFOGENE is a database of known and predicted gene structures with descriptions of basic functional signals and gene components. It provides a possibility to create compilations of sequences with a given gene feature as well as to accumulate and analyze predicted genes in finished and unfinished sequences from genome sequencing projects. Protein sequence similarity searches in the database of predicted proteins is offered through the BLASTP program. INFOGENE is realized under the Sequence Retrieval System that provides useful links with the other informational databases. The database is available through the WWW server of the Computational Genomics Group at http://genomic.sanger.ac.uk/db.html
BACKGROUND: Gut microbiota are potential sources of probiotics and play an essential role in maintaining intestinal health. Limosilactobacillus reuteri PSC102 (L. reuteri PSC102), which was isolated from the feces of healthy pigs, exhibited health-beneficial properties. AIM: We aimed to conduct a whole-genome sequencing analysis of L. reuteri PSC102 to determine its molecular characteristics as a probiotic strain. METHODS: Limosilactobacillus reuteri PSC102 cells were cultured in De Man-Rogosa-Sharpe medium, followed by DNA extraction for genomic analysis using the PacBio-Illumina sequencing platform. The EzBioCloud software was used to perform gene assembly, and the genes were interpreted by the National Center for Biotechnology Information (NCBI) and the Glimmer program. Core and pan-genomic analyses were performed to assess the extent of functional conservation in the genomic sequence. Moreover, the NCBI database and the Basic Local Alignment Search Tool software were used to identify antimicrobial resistance genes and virulence factors. RESULTS: Limosilactobacillus reuteri PSC102 consists of a single circular chromosome with 2,048,626 bp, a guanine- cytosine of 38.9%, 18 rRNA genes, and 69 tRNA genes. Among the 1,846 protein-coding sequences, genes associated with probiotic characteristics were identified, including genes involved in host-microbe interactions, stress tolerance, biogenesis, and defense mechanisms. Furthermore, the genome of L. reuteri PSC102 comprises 2,446 pan-genome and 1,222 core-genome orthologous gene clusters. A total of 74 unique genes were identified in L. reuteri PSC102 genome. These genes mostly encode proteins potentially involved in the transport and metabolism of amino acids and carbohydrates. Moreover, antibacterial resistance genes and virulence factors were absent in L. reuteri PSC102. CONCLUSION: The results of the molecular insight into L. reuteri PSC102 corroborates its use as a probiotic in humans and other animals.
Indigenous chickens play a critical role in food security and climate resilience in smallholder systems, yet their genomic diversity and adaptive potential remain insufficiently characterised. This study employed low-pass whole-genome sequencing (LP-WGS; 0.2-1.99×) to investigate genomic diversity, population structure, inbreeding and candidate environment-associated genomic variation in 33 chickens from highland, midland, and lowland agroecologies in the Tigray region of northern Ethiopia. After imputation and stringent filtering, 23.4 million high-confidence SNPs were retained, including ~ 17% novel variants, indicating substantial uncharacterised genetic diversity in these populations. SNP density (13.8 ± 8.6 SNPs/kb) was comparable to values reported from high-coverage Ethiopian chicken datasets, demonstrating the suitability of LP-WGS for population genomics in resource-limited settings. Marked differences in genomic diversity were observed among ecotypes: midland chickens showed the highest nucleotide diversity (π = 0.00267), followed by lowland (π = 0.00233), whereas highland chickens showed the lowest diversity (π = 0.00203) and elevated genomic inbreeding (FROH and FHOM ≈ 0.18). Population structure analyses revealed clear genetic separation among ecotypes. PCA (13.91% variation explained) distinguished lowland chickens along PC1 and separated highland from midland along PC2, while ADMIXTURE and FST patterns supported three major ancestral genomic backgrounds. Functional annotation of private missense variants uncovered distinct adaptive signatures reflecting the contrasting agroecological conditions. Highland chickens showed enrichment of candidate genes potentially involved in physiological processes relevant to high-altitude environments, including cold response, angiogenesis, cardiovascular regulation and metabolic homeostasis (eg., PARP1, ACOX2, ITGB3, EDNRB, SOX8, and SOX10). Midland chickens exhibited candidate signals of selection in genes with known roles in innate antiviral immunity, bacterial defence and inflammatory regulation (eg., BAK1, CLSTN1, CYSLTR1, CYSLTR2, CXCR7, GIPR, DSCAM, GDAP1, TLR3, TLR4, TLR7, IFIH1, ADORA1, EPHB1, and TMPRSS2). Lowland chickens displayed candidate variants associated with heat-stress response, DNA damage repair, oxidative balance and cardiovascular support under extreme temperatures (e.g., MLH1, BDKRB1, GPR19, FLT1, CCL18, TGM2, and RAMP3). Overall, the results indicate substantial genomic differentiation among ecotypes and suggest candidate environment-associated genetic divergence across Tigray's diverse agroecological zones. These populations may represent important reservoirs of adaptive genetic variation for climate-resilient poultry breeding, warranting further functional validation and conservation-oriented management.
GENOMES: With the DNA sequencing of most key model organisms finished or well on the way, one genomics institute is filling an important niche, using novel criteria to take on species from organisms that occupy a unique place in the evolutionary tree to others that have an ability to digest explosives.
In addition to their value for wood products, members of the genus Populus (poplars) provide a range of ecological services, including carbon sequestration, bioremediation, nutrient cycling, biofiltration and diverse habitats. They are also widely used model organisms for tree molecular biology and biotechnology. The sequencing of the poplar genome to an approximately 6x depth adds to a long list of important attributes for research. These include facile transformation, vegetative propagation, rapid growth, modest genome size and extensive expressed sequence tags. Here, we discuss how the genome sequence and transformability of poplar, together with its high levels of genetic and ecological diversity, are enabling new insights into the genetic programs controlling ontogeny, ecological adaptation and environmental physiology of trees.
It has been more than 10 years since the first bacterial genome sequence was published. Hundreds of bacterial genome sequences are now available for comparative genomics, and searching a given protein against more than a thousand genomes will soon be possible. The subject of this review will address a relatively straightforward question: "What have we learned from this vast amount of new genomic data?" Perhaps one of the most important lessons has been that genetic diversity, at the level of large-scale variation amongst even genomes of the same species, is far greater than was thought. The classical textbook view of evolution relying on the relatively slow accumulation of mutational events at the level of individual bases scattered throughout the genome has changed. One of the most obvious conclusions from examining the sequences from several hundred bacterial genomes is the enormous amount of diversity--even in different genomes from the same bacterial species. This diversity is generated by a variety of mechanisms, including mobile genetic elements and bacteriophages. An examination of the 20 Escherichia coli genomes sequenced so far dramatically illustrates this, with the genome size ranging from 4.6 to 5.5 Mbp; much of the variation appears to be of phage origin. This review also addresses mobile genetic elements, including pathogenicity islands and the structure of transposable elements. There are at least 20 different methods available to compare bacterial genomes. Metagenomics offers the chance to study genomic sequences found in ecosystems, including genomes of species that are difficult to culture. It has become clear that a genome sequence represents more than just a collection of gene sequences for an organism and that information concerning the environment and growth conditions for the organism are important for interpretation of the genomic data. The newly proposed Minimal Information about a Genome Sequence standard has been developed to obtain this information.
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Genome sequencing currently requires DNA from pools of numerous nearly identical cells (clones), leaving the genome sequences of many difficult-to-culture microorganisms unattainable. We report a sequencing strategy that eliminates culturing of microorganisms by using real-time isothermal amplification to form polymerase clones (plones) from the DNA of single cells. Two Escherichia coli plones, analyzed by Affymetrix chip hybridization, demonstrate that plonal amplification is specific and the bias is randomly distributed. Whole-genome shotgun sequencing of Prochlorococcus MIT9312 plones showed 62% coverage of the genome from one plone at a sequencing depth of 3.5x, and 66% coverage from a second plone at a depth of 4.7x. Genomic regions not revealed in the initial round of sequencing are recovered by sequencing PCR amplicons derived from plonal DNA. The mutation rate in single-cell amplification is <2 x 10(5), better than that of current genome sequencing standards. Polymerase cloning should provide a critical tool for systematic characterization of genome diversity in the biosphere.
As sequenced genomes become larger and sequencing process becomes faster, there is a need to develop a tool to analyze sequences in the whole genomic scale. However, on-memory algorithms such as suffix tree and suffix array are not applicable to the analysis of whole genome sequence set, since the size of individual whole genome ranges from several million base pairs to hundreds billion base pairs. In order to effectively manipulate the huge sequence data, it is necessary to use the indexed data structure for external memory. In this paper, we introduce a workbench called SequeX for the analysis and visualization of whole genome sequences using SSB-tree (Static SB-tree). It consists of two parts: the analysis query subsystem and the visualization subsystem. The query subsystem supports various transactions such as pattern matching, k-occurrence, and k-mer analysis. The visualization subsystem helps biologists to easily understand whole genome structure and feature by sequence viewer, annotation viewer, CGR (Chaos Game Representation) viewer, and k-mer viewer. The system also supports a user-friendly programming interface based on Java script for batch processing and the extension for a specific purpose of a user. SequeX can be used to identify conserved genes or sequences by the analysis of the common k-mers and annotation. We analyze the common k-mer for 72 microbial genomes announced by Entrez, and find an interesting biological fact that the longest common k-mer for 72 sequences is 11-mer, and only 11 such sequences exist. Finally we note that many common k-mers occur in conserved region such as CDS, rRNA, and tRNA.
The genome sequence database (GSDB) is a complete, publicly available relational database of DNA sequences and annotation maintained by the National Center for Genome Resources (NCGR) under a Cooperative Agreement with the US Department of Energy (DOE). GSDB provides direct, client- server access to the database for data contributions, community annotation and SQL queries. The GSDB Annotator, a multi-platform graphic user interface, is freely available. Automatically updated relational replicates of GSDB are also freely available.
We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress.
The functional analysis of sequenced genomes will be facilitated by the development of tools for the rapid mapping of mutations. We have developed a systematic approach to genetic mapping in Caulobacter crescentus that is based on bacteriophage-mediated transduction of strategically placed antibiotic resistance markers. The genomic DNA sequence was used to identify sites distributed evenly around the chromosome at which plasmids could be nondisruptively integrated. DNA fragments from these sites were amplified by PCR and cloned into a kanamycin-resistant (Kan(r)) suicide vector. Delivery of these plasmids into C. crescentus resulted in integration via homologous recombination. A set of 41 strains containing Kan(r) markers at 100-kb intervals was thereby generated. These strains serve as donors for generalized transduction using bacteriophage phiCr30, which can transduce at least 120 kb of DNA. Transductants are selected with kanamycin and screened for loss of the mutant phenotype to assess linkage between the marker and the site of the mutation. The dependence of cotransduction frequency on sequence distance was evaluated using several markers and mutant strains. With these data as a standard, previously unmapped mutations were readily localized to DNA sequence intervals equivalent to less than 1% of the genome. Candidate genes within the interval were then examined further by subcloning and complementation analysis. Mutations resulting in sensitivity to ampicillin, in nutritional auxotrophies, or temperature-sensitive growth were mapped. This approach to genetic mapping should be applicable to other bacteria with sequenced genomes for which generalized transducing phage are available.
We have developed a streamlined, reproducible method for performing genomic chemical sequencing reactions on the genomic DNA of Mycoplasma capricolum, which has a genome size of about 750,000 base pairs and whose composition is 75% AT. The general modifications that ensure reproducibility and allow the processing of multiple samples can be widely adopted to other large-scale sequencing projects, while the specific modifications to the chemical reactions are applicable to the sequencing of other DNAs with a high AT content.