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PubMed2005

Antigenome technology: a novel approach for the selection of bacterial vaccine candidate antigens.

A novel approach for the identification of protein antigens from bacterial pathogens was previously developed in our laboratory that combines the advantages of full genome coverage and serological antigen identification. We have applied this technology to several bacterial pathogens of the genera Staphylococcus and Streptococcus and have, as a result, defined the "antigenome" of these pathogens. This catalogue defines the most relevant antigenic proteins that are targeted by the human immune system, including their antibody binding sites. The antigenome technology offers an integrated approach for antigen validation in order to select the most promising candidates for the development of subunit vaccines against the targeted bacterial diseases. Using this technology, novel protective antigens were discovered from several important human pathogens.

Animals↗

PubMed2005

[Antigenome technology: rapid and comprehensive identification and validation of antigens as putative components in vaccines against bacterial pathogens].

The resolution of complete genome sequences of many pathogens has dramatically expanded our perspectives in developing novel vaccines by increasing both the speed and scale of target identification. Genomics-based technologies with their significant advantages can be applied to most pathogens and have exploited genome sequence information in alliance with adjunct technologies, including bioinformatics, expression analyses (microarrays, in vivo expression technologies), random mutagenesis or protein/peptide based selection methods (proteomics and immunoselection using expression libraries). A novel approach that combines the advantages of full genome coverage and serological antigen identification has been developed in the laboratory of the authors for the identification of protein antigens from bacterial pathogens. This technology has been applied to several bacterial pathogens and, as a result, defined the most relevant antigenic proteins that are targeted by the human immune system, including their antibody binding sites (epitopes). This catalogue of antigens is termed the antigenome and the novel technology offers an integrated approach for antigen validation in order to select the most promising candidates for the development of subunit vaccines against the targeted bacterial diseases. Using the antigenome technology, novel protective antigens had been discovered from several important human pathogens.

Antigens, Bacterial↗

PubMed2026

Library strategies differentially shape microbial, functional, and host signals in clinical metagenomic sequencing.

Metagenomic next-generation sequencing (mNGS) is increasingly used in infectious disease diagnostics, yet how library preparation shapes the microbial, functional, and host signals recovered from clinical samples remains poorly defined. Here, we performed a within-sample parallel comparison of three mNGS library preparation strategies-DNA-based libraries (DNAlib), RNA-based libraries (RNAlib), and total nucleic acid-based libraries (TNAlib)-across a diverse range of clinical specimens spanning five sample types. Using a curated clinical infectome as a benchmark, we show that library strategies are not interchangeable but capture distinct biological dimensions of the same specimen. RNAlib provided the most comprehensive standalone recovery of the clinical infectome, with improved detection of RNA viruses and cellular pathogens, enhanced resolution of resistance and virulence signals, and preservation of infection-associated host immune signatures. DNAlib showed stronger baseline recovery of DNA viruses and broader host genome coverage, whereas the TNAlib workflow evaluated here largely behaved as an intermediate strategy rather than a consistent improvement over dedicated DNA- or RNA-based workflows. Together, these results establish that the library preparation protocol is a major determinant of how clinical mNGS data should be interpreted and provide a framework for selecting sequencing strategies according to specific diagnostic and biological questions.IMPORTANCEMetagenomic sequencing is increasingly used in infectious disease research and clinical diagnostics, but different library preparation strategies may recover fundamentally different biological signals from the same sample. These signals include not only pathogens but also background microbes, microbial functional activity, and host immune-response patterns. Here, we systematically compared DNA-, RNA-, and total nucleic acid-based metagenomic sequencing libraries using the same clinical samples processed in parallel. We found that the three strategies did not provide equivalent information. RNA-based sequencing generated the most informative single-library view of infection, particularly for RNA viruses, cellular pathogens, functional microbial signals, and host immune-response patterns. DNA-based sequencing was more effective for DNA virus and host genome recovery, whereas the total nucleic acid sequencing workflow evaluated here generally behaved as an intermediate strategy. These findings show that library preparation can substantially influence the interpretation of metagenomic data.

functional characterization↗

PubMed2001

An enhanced linkage map of the sheep genome comprising more than 1000 loci.

A medium-density linkage map of the ovine genome has been developed. Marker data for 550 new loci were generated and merged with the previous sheep linkage map. The new map comprises 1093 markers representing 1062 unique loci (941 anonymous loci, 121 genes) and spans 3500 cM (sex-averaged) for the autosomes and 132 cM (female) on the X chromosome. There is an average spacing of 3.4 cM between autosomal loci and 8.3 cM between highly polymorphic [polymorphic information content (PIC) > or = 0.7] autosomal loci. The largest gap between markers is 32.5 cM, and the number of gaps of > 20 cM between loci, or regions where loci are missing from chromosome ends, has been reduced from 40 in the previous map to 6. Five hundred and seventy-three of the loci can be ordered on a framework map with odds of > 1000 : 1. The sheep linkage map contains strong links to both the cattle and goat maps. Five hundred and seventy-two of the loci positioned on the sheep linkage map have also been mapped by linkage analysis in cattle, and 209 of the loci mapped on the sheep linkage map have also been placed on the goat linkage map. Inspection of ruminant linkage maps indicates that the genomic coverage by the current sheep linkage map is comparable to that of the available cattle maps. The sheep map provides a valuable resource to the international sheep, cattle, and goat gene mapping community.

Animals↗

PubMed2002

Identification of novel hrp-regulated genes through functional genomic analysis of the Pseudomonas syringae pv. tomato DC3000 genome.

Pseudomonas syringae pv. tomato (Pst) strain DC3000 infects the model plants Arabidopsis thaliana and tomato, causing disease symptoms characterized by necrotic lesions surrounded by chlorosis. One mechanism used by Pst DC3000 to infect host plants is the type III protein secretion system, which is thought to deliver multiple effector proteins to the plant cell. The exact number of type III effectors in Pst DC3000 or any other plant pathogenic bacterium is not known. All known type III effector genes of P. syringae are regulated by HrpS, an NtrC family protein, and the HrpL alternative sigma factor, which presumably binds to a conserved cis element (called the "hrp box") in the promoters of type III secretion-associated genes. In this study, we designed a search motif based on the promoter sequences conserved in 12 published hrp operons and putative effector genes in Pst DC3000. Seventy-three predicted genes were retrieved from the January 2001 release of the Pst DC3000 genome sequence, which had 95% genome coverage. The expression of the 73 genes was analysed by microarray and Northern blotting, revealing 24 genes/operons (including eight novel genes), the expression of which was consistently higher in hrp-inducing minimal medium than in nutrient-rich Luria-Bertani broth. Expression of all eight genes was dependent on the hrpS gene. Most were also dependent on the hrpL gene, but at least one was dependent on the hrpS gene, but not on the hrpL gene. An AvrRpt2-based type III translocation assay provides evidence that some of the hrpS-regulated novel genes encode putative effector proteins.

Arabidopsis↗

PubMed2006

Whole genome expression profiling of the medial and lateral substantia nigra in Parkinson's disease.

We have used brain tissue from clinically well-documented and neuropathologically confirmed cases of sporadic Parkinson's disease to establish the transcriptomic expression profile of the medial and lateral substantia nigra. In addition, the superior frontal cortex was analyzed in a subset of the same cases. DNA oligonucleotide microarrays were employed, which provide whole human genome coverage. A total of 570 genes were found to be differentially regulated at a high level of significance. A large number of differentially regulated expressed sequence tags were also identified. Levels of mRNA sequences encoded by genes of key interest were validated by means of quantitative real-time polymerase chain reaction (PCR). Comparing three different normalization procedures, results based on the recently published GeneChip Robust Multi Array algorithm were found to be the most accurate predictor of real-time PCR results. Several new candidate genes which map to PARK loci are reported. In addition, the DNAJ family of chaperones is discussed in the context of Parkinson's disease pathogenesis.

Aged↗

PubMed2006

Genomic organization and expression analyses of putrescine pathway genes in soybean.

Putrescine is synthesized using one of two alternative pathways in plants, from arginine by arginine decarboxylase (ADC) or from ornithine by ornithine decarboxylase (ODC) and is catabolized by diamine oxidase (DAO). A survey of approximately 310,000 expressed sequenced tags (ESTs) in soybean EST libraries identified diverse representation of ADC, ODC, and DAO ESTs, with ODC being least frequent and DAO ESTs most abundant. Southern analysis suggested that ADC and ODC belong to small gene families, and DAO is the most divergent. Using three bacterial artificial chromosome (BAC) libraries, 26X genome equivalents, two common loci for ADC and DAO and one independent DAO locus were identified. ADC and DAO are physically linked in the soybean genome within approximately 150 kb. Identification of genomic regions encoding ODC proved difficult and required using additional BAC libraries, increasing genome coverage to approximately 40X. Using Real Time reverse transcriptase-polymerase chain reaction (RT-PCR), higher steady-state levels of ADC than ODC in roots, leaves, shoot apices, and dry seeds suggested that ADC is the predominant pathway for putrescine biosynthesis in soybean. However, organ-specific expression showed that root is the major site of ODC transcription. Significantly elevated accumulation of ADC mRNA and elevated putrescine content in seeds of the fasciation mutant compared with the wild type may stimulate cell divisions and establishment of enlarged apical meristem during early mutant ontogeny. The DAO frequent representation in EST libraries constructed from root tissue and elevated steady-state levels in roots compared to above ground tissues show DAO is critical for regulation of putrescine content in soybean roots.

Amine Oxidase (Copper-Containing)↗

PubMedDate not supplied

A male linkage map of the cattle (Bos taurus) genome.

A male linkage map of the cattle (Bos taurus) genome was constructed using nine large half-sib families. The map consists of 269 loci, of which 249 are microsatellites and 20 are structural genes. Among the 249 microsatellites, 140 are markers selected from other maps and 98 are new assignments. Chromosome assignment were established for 35 new markers by somatic cell hybrid analysis, of which 26 were confirmed by linkage analysis. Genome coverage is 1975 cM contained within terminal markers on all 29 autosomes. The average distance between adjacent loci is 9.7 cM, with 72.1% of the map intervals < or = 15 cM and 4.9% of the intervals > or = 25 cM. The inclusion of mapped markers permitted integration and comparisons with other maps, facilitating the identification of discrepancies in chromosome assignment, gene order, and map distance. The inclusion of Type I and blood group markers in the map was useful for comparative mapping, revealing possible blood group orthologies between humans and cattle. The map generated will serve as a useful tool for comparative mapping, mapping of quantitative trait loci and marker assisted selection.

Animals↗

PubMed2003

Mapping of quantitative trait loci controlling trypanotolerance in a cross of tolerant West African N'Dama and susceptible East African Boran cattle.

Trypanosomosis, or sleeping sickness, is a major disease constraint on livestock productivity in sub-Saharan Africa. To identify quantitative trait loci (QTL) controlling resistance to trypanosomosis in cattle, an experimental cross was made between trypanotolerant African N'Dama (Bos taurus) and trypanosusceptible improved Kenya Boran (Bos indicus) cattle. Sixteen phenotypic traits were defined describing anemia, body weight, and parasitemia. One hundred seventy-seven F2 animals and their parents and grandparents were genotyped at 477 molecular marker loci covering all 29 cattle autosomes. Total genome coverage was 82%. Putative QTL were mapped to 18 autosomes at a genomewise false discovery rate of <0.20. The results are consistent with a single QTL on 17 chromosomes and two QTL on BTA16. Individual QTL effects ranged from approximately 6% to 20% of the phenotypic variance of the trait. Excluding chromosomes with ambiguous or nontrypanotolerance effects, the allele for resistance to trypanosomosis originated from the N'Dama parent at nine QTL and from the Kenya Boran at five QTL, and at four QTL there is evidence of an overdominant mode of inheritance. These results suggest that selection for trypanotolerance within an F2 cross between N'Dama and Boran cattle could produce a synthetic breed with higher trypanotolerance levels than currently exist in the parental breeds.

Anemia↗

PubMed1999

A genome scan for QTL influencing milk production and health traits in dairy cattle.

A genome scan was conducted in the North American Holstein-Friesian population for quantitative trait loci (QTL) affecting production and health traits using the granddaughter design. Resource families consisted of 1,068 sons of eight elite sires. Genome coverage was estimated to be 2,551 cM (85%) for 174 genotyped markers. Each marker was tested for effects on milk yield, fat yield, protein yield, fat percentage, protein percentage, somatic cell score, and productive herd life using analysis of variance. Joint analysis of all families identified marker effects on 11 chromosomes that exceeded the genomewide, suggestive, or nominal significance threshold for QTL effects. Large marker effects on fat percentage were found on chromosomes 3 and 14, and multimarker regression analysis was used to refine the position of these QTL. Half-sibling families from Israeli Holstein dairy herds were used in a daughter design to confirm the presence of the QTL for fat percentage on chromosome 14. The QTL identified in this study may be useful for marker-assisted selection and for selection of a refined set of candidate genes affecting these traits.

Animals↗

PubMed2003

A graph-theoretic approach to comparing and integrating genetic, physical and sequence-based maps.

For many species, multiple maps are available, often constructed independently by different research groups using different sets of markers and different source material. Integration of these maps provides a higher density of markers and greater genome coverage than is possible using a single study. In this article, we describe a novel approach to comparing and integrating maps by using abstract graphs. A map is modeled as a directed graph in which nodes represent mapped markers and edges define the order of adjacent markers. Independently constructed graphs representing corresponding maps from different studies are merged on the basis of their common loci. Absence of a path between two nodes indicates that their order is undetermined. A cycle indicates inconsistency among the mapping studies with regard to the order of the loci involved. The integrated graph thus produced represents a complete picture of all of the mapping studies that comprise it, including all of the ambiguities and inconsistencies among them. The objective of this representation is to guide additional research aimed at interpreting these ambiguities and inconsistencies in locus order rather than presenting a "consensus order" that ignores these problems.

Chromosome Mapping↗

PubMed1999

A high-density physical map of Sinorhizobium meliloti 1021 chromosome derived from bacterial artificial chromosome library.

As part of the European Sinorhizobium meliloti (strain 1021) chromosome sequencing project, four genomic bacterial artificial chromosome (BAC) libraries have been constructed, one of which was mainly used for chromosome mapping. This library consists of 1,824 clones with an average insert size of 80 kilobases and represents approximately 20-fold total genome coverage [6.8 megabases (Mbs)]. PCR screening of 384 BAC clones with 447 chromosomal markers (PCR primer pairs), consisting of 73 markers representing 118 genes (40 individual genes and 78 genes clustered in 23 operons), two markers from the rrn operon (three loci), four markers from insertion sequences (approximately 16 loci) and 368 sequence-tagged sites allowed the identification of 252 chromosomal BAC clones and the construction of a high-density physical map of the whole 3.7-Mb chromosome of S. meliloti. An average of 5.5 overlapping and colinear BAC clones per marker, correlated with a low rate of deleted or rearranged clones (0.8%) indicate a solid BAC contigation and a correct mapping. Systematic BLASTX analysis of sequence-tagged site marker sequences allowed prediction of a biological function for a number of putative ORFs. Results are available at. This map, whose resolution averages one marker every 9 kilobases, should provide a valuable tool for further sequencing, functional analysis, and positional cloning.

Chromosome Mapping↗

PubMed2025

Coronavirus Cryptic Landscape and Draft Genome of a Novel CoV Clade Related to MERS From Bats Circulating in Northeastern Brazil.

We identified seven distinct coronaviruses (CoVs) in bats from Brazil, classified into 229E-related (Alpha-CoV), Nobecovirus, Sarbecovirus, and Merbecovirus (Beta-CoV), including one closely related to MERS-like CoV with 82.8% genome coverage. To accomplish this, we screened 423 oral and rectal swabs from 16 different bat species using molecular assays, RNA sequencing, and evolutionary analysis. Notably, gaps in the spike-encoding gene led us to design new primers and perform Sanger sequencing, which revealed high similarities to MERS-related (MERSr) CoV strains found in humans and camels. Additionally, we identified key residues in the receptor-binding domain (RBD) of the spike protein, suggesting potential interactions with DPP4, the receptor for MERSr-CoV. Our analyses also revealed evidence of recombination involving our laboratory-produced sequences. These findings highlight the extensive genetic diversity of CoVs, the presence of novel viral lineages, and the occurrence of recombination events among bat CoVs circulating in Brazil, underscoring the critical role bats play as reservoirs for emerging viruses and emphasizing the necessity of ongoing surveillance to monitor the public health risks associated with CoV spillover events.

Chiroptera↗

PubMed2026

PScnv: personalized self-normalizing CNV detection with a hierarchical multi-phase framework.

MOTIVATION: Accurate detection of copy number variations (CNVs) from targeted panel sequencing remains challenging due to limited genomic coverage and pronounced sample-specific biases. Existing normalization strategies, including baseline-cohort, matched-control, and single-sample approaches, often struggle to balance noise suppression with adaptability, leading to inconsistent performance across heterogeneous samples. RESULTS: We present PScnv, a personalized self-normalizing framework for robust CNV detection from panel sequencing data. PScnv integrates a pre-built panel-of-normals (PoN) with sample-intrinsic stable chromosomes through ridge-regression normalization to generate individualized log2 ratio profiles with reduced systematic variation. CNVs are then identified using a hierarchical multi-phase segmentation pipeline incorporating z-score pre-partitioning, kernel-based correction, and circular binary segmentation. In 139 clinical tumor samples with orthogonal FISH validation at MET, ERBB2, and MTAP, PScnv showed improved accuracy and robustness over existing methods that do not require patient-matched normal samples, provided that a pre-built PoN cohort is available. AVAILABILITY: Source code is available for academic use at https://github.com/lvws/PScnv.

DNA Copy Number Variations↗

PubMed2004

Selection and characterization of conditionally active promoters in Lactobacillus plantarum, using alanine racemase as a promoter probe.

This paper describes the use of the alr gene, encoding alanine racemase, as a promoter-screening tool for the identification of conditional promoters in Lactobacillus plantarum. Random fragments of the L. plantarum WCFS1 genome were cloned upstream of the promoterless alr gene of Lactococcus lactis in a low-copy-number plasmid vector. The resulting plasmid library was introduced into an L. plantarum Deltaalr strain (MD007), and 40,000 clones were selected. The genome coverage of the library was estimated to be 98%, based on nucleotide insert sequence and restriction analyses of the inserts of randomly selected clones. The library was screened for clones that were capable of complementing the D-alanine auxotroph phenotype of MD007 in media containing up to 10, 100, or 300 micro g of the competitive Alr inhibitor D-cycloserine per ml. Western blot analysis with polyclonal antibodies raised against lactococcal Alr revealed that the Alr production level required for growth increased in the presence of increasing concentrations of D-cycloserine, adding a quantitative factor to the primarily qualitative nature of the alr complementation screen. Screening of the alr complementation library for clones that could grow only in the presence of 0.8 M NaCl resulted in the identification of eight clones that upon Western blot analysis showed significantly higher Alr production under high-salt conditions than under low-salt conditions. These results established the effectiveness of the alanine racemase complementation screening method for the identification of promoters on their conditional or constitutive activity.

Alanine Racemase↗

PubMed2006

Multiple displacement amplification to create a long-lasting source of DNA for genetic studies.

In many situations there may not be sufficient DNA collected from patient or population cohorts to meet the requirements of genome-wide analysis of SNPs, genomic copy number polymorphisms, or acquired copy number alternations. When the amount of available DNA for genotype analysis is limited, high performance whole-genome amplification (WGA) represents a new development in genetic analysis. It is especially useful for analysis of DNA extracted from stored histology slides, tissue samples, buccal swabs, or blood stains collected on filter paper. The multiple displacement amplification (MDA) method, which relies on isothermal amplification using the DNA polymerase of the bacteriophage phi29, is a recently developed technique for high performance WGA. This review addresses new trends in the technical performance of MDA and its applications to genetic analyses. The main challenge of WGA methods is to obtain balanced and faithful replication of all chromosomal regions without the loss of or preferential amplification of any genomic loci or allele. In multiple comparisons to other WGA methods, MDA appears to be most reliable for genotyping, with the most favorable call rates, best genomic coverage, and lowest amplification bias.

DNA↗

PubMed2006

2-D protein maps of rat gastrocnemius and soleus muscles: a tool for muscle plasticity assessment.

Functional characterization of muscle fibers relies on ATPase activity and on differential measurements of metabolic proteins, including mitochondrial and glycolytic enzymes, glucose, lactate and lactic acid transporters, calcium cycling proteins and components of the contractile machinery. The recent introduction of microarray technology has enabled detailed gene expression studies under different physiological and pathological conditions, thus generating novel hypotheses on muscle function. However, microarray approaches are limited by the incomplete genome coverage of currently available chips, and by poor correlation between mRNA concentration and protein expression level. We have used 2-DE and MS to build a reference map of proteins from rat mixed gastrocnemius and soleus muscle, and to assess qualitative and quantitative differences in protein distribution between these two functionally dissimilar muscles. More than 800 spots on each gel were detected by silver staining, of which 167 were excised, digested in-gel with trypsin and analyzed by ESI-MS/MS. One hundred and twenty eight distinct gene products were identified, including metabolic, transport and contractile proteins. Forty one spots displayed differences in relative expression level between mixed gastrocnemius and soleus samples. These data not only enable differentiation of functionally distinct slow-twitch and fast-twitch fiber types, but also provide tools for investigating muscle plasticity in response to physiological and environmental conditions such as aging or hypoxia.

Aging↗

PubMed2001

Rat-mouse and rat-human comparative maps based on gene homology and high-resolution zoo-FISH.

The laboratory rat, Rattus norvegicus, and the laboratory mouse, Mus musculus, are key animal models in biomedical research. A deeper understanding of the genetic interrelationsships between Homo sapiens and these two rodent species is desirable for extending the usefulness of the animal models. We present comprehensive rat-human and rat-mouse comparative maps, based on 1090 gene homology assignments available for rat genes. Radiation hybrid, FISH, and zoo-FISH mapping data have been integrated to produce comparative maps that are estimated to comprise 83-100% of the conserved regions between rat and mouse and 66-82% of the conserved regions between rat and human. The rat-mouse zoo-FISH analysis, supported by data for individual genes, revealed nine previously undetected conserved regions compared to earlier reports. Since there is almost complete genome coverage in the rat-mouse comparative map, we conclude that it is feasible to make accurate predictions of gene positions in the rat based on gene locations in the mouse.

Animals↗