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PubMed2006

Determination and analysis of the DNA sequence of highly attenuated herpes simplex virus type 1 mutant HF10, a potential oncolytic virus.

A spontaneously occurring herpes simplex virus type 1 (HSV-1) mutant, designated HF10, replicates very efficiently and induces extensive cell fusion in most transformed cells as well as Vero cells, but is highly attenuated in mice when inoculated by peripheral routes of infection. Recent studies have shown that HF10 is a promising agent for use in oncolytic virotherapy. In this study, we sequenced the genome of HF10 and compared it with that of HSV-1 strain 17, a reference strain with the syn+ phenotype. The sequencing covered whole regions corresponding to all open reading frames of strain 17, and the overall putative amino acid identity between HF10 and strain 17 was 99.1% except for proteins encoded by three genes with frame-shift mutations. HF10 had a number of deletions and insertions in the genome, resulting in the lack of the functional expression of UL43, UL49.5, UL55, UL56 and latency-associated transcripts. Additionally, HF10 had amino acid changes in genes involved in the regulation of syncytium formation, including UL1, UL20, UL22, UL24, UL27 and UL53. The proteins encoded by UL1, UL2, UL11, UL44, US1, US7, US8.5, US10 and US12 exhibited a relatively high divergence. These data provide the genetic background of HF10 and insight into the molecular mechanism of HSV-1 replication and pathogenicity.

Amino Acid Substitution↗
PubMed2005

Functional coverage of the human genome by existing structures, structural genomics targets, and homology models.

The bias in protein structure and function space resulting from experimental limitations and targeting of particular functional classes of proteins by structural biologists has long been recognized, but never continuously quantified. Using the Enzyme Commission and the Gene Ontology classifications as a reference frame, and integrating structure data from the Protein Data Bank (PDB), target sequences from the structural genomics projects, structure homology derived from the SUPERFAMILY database, and genome annotations from Ensembl and NCBI, we provide a quantified view, both at the domain and whole-protein levels, of the current and projected coverage of protein structure and function space relative to the human genome. Protein structures currently provide at least one domain that covers 37% of the functional classes identified in the genome; whole structure coverage exists for 25% of the genome. If all the structural genomics targets were solved (twice the current number of structures in the PDB), it is estimated that structures of one domain would cover 69% of the functional classes identified and complete structure coverage would be 44%. Homology models from existing experimental structures extend the 37% coverage to 56% of the genome as single domains and 25% to 31% for complete structures. Coverage from homology models is not evenly distributed by protein family, reflecting differing degrees of sequence and structure divergence within families. While these data provide coverage, conversely, they also systematically highlight functional classes of proteins for which structures should be determined. Current key functional families without structure representation are highlighted here; updated information on the "most wanted list" that should be solved is available on a weekly basis from http://function.rcsb.org:8080/pdb/function_distribution/index.html.

Databases, Protein↗
PubMed1992

Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt.

We have developed a restriction map of the chromosome 21 breakpoint region involved in t(8;21)(q22;q22.3) acute myelogenous leukemia (AML) and have isolated a genomic junction clone containing chromosome 8 and 21 material. Using probes from these regions, rearrangements have been identified in each of nine cases of t(8;21) AML examined. In addition, we have isolated cDNA clones from a t(8;21) AML cDNA library that contain fused sequences from chromosome 8 and 21. The chromosome 8 component, referred to as ETO (for eight twenty-one), is encoded over a large genomic region, as suggested by the analysis of corresponding yeast artificial chromosomes (YACs). The DNA sequence of the chromosome 21 portion of the fusion transcript is derived from the normal AML1 gene. A striking similarity (67% identity over 387 bp, with a corresponding 69% amino acid identity) was detected between AML1 and the Drosophila segmentation gene, runt. The critical consequence of the translocation is the juxtaposition of 5' sequences of AML1 to 3' sequences of ETO, oriented telomere to centromere on the der(8) chromosome.

Amino Acid Sequence↗
PubMed2001

iProClass: an integrated, comprehensive and annotated protein classification database.

The iProClass database is an integrated resource that provides comprehensive family relationships and structural and functional features of proteins, with rich links to various databases. It is extended from ProClass, a protein family database that integrates PIR superfamilies and PROSITE motifs. The iProClass currently consists of more than 200,000 non-redundant PIR and SWISS-PROT proteins organized with more than 28,000 superfamilies, 2600 domains, 1300 motifs, 280 post-translational modification sites and links to more than 30 databases of protein families, structures, functions, genes, genomes, literature and taxonomy. Protein and family summary reports provide rich annotations, including membership information with length, taxonomy and keyword statistics, full family relationships, comprehensive enzyme and PDB cross-references and graphical feature display. The database facilitates classification-driven annotation for protein sequence databases and complete genomes, and supports structural and functional genomic research. The iProClass is implemented in Oracle 8i object-relational system and available for sequence search and report retrieval at http://pir.georgetown.edu/iproclass/.

Databases, Factual↗
PubMed2024

WHOLE GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are protozoan parasites that cause severe illness in vulnerable human populations. Obtaining pure Cryptosporidium DNA from clinical and environmental samples is challenging because the oocysts shed in contaminated feces are limited in quantity, difficult to purify efficiently, may derive from multiple species, and yield limited DNA (<40 fg/oocyst). Here, we develop and validate a set of 100,000 RNA baits (CryptoCap_100k) based on six human-infecting Cryptosporidium spp. (C. cuniculus, C. hominis, C. meleagridis, C. parvum, C. tyzzeri, and C. viatorum) to enrich Cryptosporidium spp. DNA from a wide array of samples. We demonstrate that CryptoCap_100k increases the percentage of reads mapping to target Cryptosporidium references in a wide variety of scenarios, increasing the depth and breadth of genome coverage, facilitating increased accuracy of detecting and analyzing species within a given sample, while simultaneously decreasing costs, thereby opening new opportunities to understand the complex biology of these important pathogens.

Journal Article↗
PubMed2001

A review of the DNA standard reference materials developed by the National Institute of Standards and Technology.

The Standard Reference Materials Program at the US National Institute of Standards and Technology (NIST) has three human DNA standard reference materials (SRM 2390, SRM 2391a, and SRM 2392) currently available [1, 2]. Both the DNA profiling SRM 2390 and the polymerase chain reaction (PCR)-based DNA profiling SRM 2391a are intended for use in forensic and paternity identifications, for instructional law enforcement, or for non-clinical research purposes and are not intended for clinical diagnostics. The mitochondrial DNA (mtDNA) SRM 2392 is to provide standardization and quality control when performing PCR and sequencing any segment or the entire 16,569 base pairs that comprise human mitochondrial DNA. SRM 2392 is designed for use by the forensic, medical, and toxicological communities for human identification, disease diagnosis or mutation detection.

DNA↗
PubMed1996

MITOMAP: a human mitochondrial genome database.

We have developed a comprehensive database (MITOMAP) for the human mitochondrial DNA (mtDNA), the first component of the human genome to be completely sequenced [Anderson et al. (1981) Nature 290, 457-465]. MITOMAP uses the mtDNA sequence as the unifying element for bringing together information on mitochondrial genome structure and function, pathogenic mutations and their clinical characteristics, population associated variation, and gene- gene interactions. As increasingly larger regions of the human genome are sequenced and characterized, the need for integrating such information will grow. Consequently, MITOMAP not only provides a valuable reference for the mitochondrial biologist, it may also provide a model for the development of information storage and retrieval systems for other components of the human genome.

Amino Acid Sequence↗
PubMed2025

Analysis of targeted and whole genome sequencing of PacBio HiFi reads for a comprehensive genotyping of gene-proximal and phenotype-associated Variable Number Tandem Repeats.

Variable Number Tandem repeats (VNTRs) refer to repeating motifs of size greater than five bp. VNTRs are an important source of genetic variation, and have been associated with multiple Mendelian and complex phenotypes. However, the highly repetitive structures require reads to span the region for accurate genotyping. Pacific Biosciences HiFi sequencing spans large regions and is highly accurate but relatively expensive. Therefore, targeted sequencing approaches coupled with long-read sequencing have been proposed to improve efficiency and throughput. In this paper, we systematically explored the trade-off between targeted and whole genome HiFi sequencing for genotyping VNTRs. We curated a set of 10&#xa0;,&#xa0;787 gene-proximal (G-)VNTRs, and 48 phenotype-associated (P-)VNTRs of interest. Illumina reads only spanned 46% of the G-VNTRs and 71% of P-VNTRs, motivating the use of HiFi sequencing. We performed targeted sequencing with hybridization by designing custom probes for 9,999 VNTRs and sequenced 8 samples using HiFi and Illumina sequencing, followed by adVNTR genotyping. We compared these results against HiFi whole genome sequencing (WGS) data from 28 samples in the Human Pangenome Reference Consortium (HPRC). With the targeted approach only 4,091 (41%) G-VNTRs and only 4 (8%) of P-VNTRs were spanned with at least 15 reads. A smaller subset of 3,579 (36%) G-VNTRs had higher median coverage of at least 63 spanning reads. The spanning behavior was consistent across all 8 samples. Among 5,638 VNTRs with low-coverage (&#xa0;<&#xa0;15), 67% were located within GC-rich regions (&#xa0;>&#xa0;60%). In contrast, the 40X WGS HiFi dataset spanned 98% of all VNTRs and 49 (98%) of P-VNTRs with at least 15 spanning reads, albeit with lower coverage. Spanning reads were sufficient for accurate genotyping in both cases. Our findings demonstrate that targeted sequencing provides consistently high coverage for a small subset of low-GC VNTRs, but WGS is more effective for broad and sufficient sampling of a large number of VNTRs.

Minisatellite Repeats↗
PubMedDate not supplied

New microsatellite markers on the linkage map of the chicken genome.

Ten polymorphic microsatellite markers mainly of the poly (TG) type were examined on the two international reference families, the East Lansing reference family and the Compton reference family. Three of the markers were polymorphic on the East Lansing family (linkage groups E), three on the Compton family (linkage groups C), and four were polymorphic on both families. The microsatellite markers were well dispersed among the various linkage groups, and there was no evidence for clustering of the markers within the map. Common microsatellite markers were able to associate linkage groups from the two genetic maps. Association of linkage groups E2 with C4, E10 with C13, and E1 with C15 are found. Linkage group E1 is located on chromosome 1, and because of the association between E1 and C15, C15 is therefore assigned to chromosome 1. A newly defined linkage group (E20) was found in case of microsatellite marker MCW14. The relative distances between markers and the gene order of the markers can change upon addition of more markers to the linkage maps.

Animals↗
PubMed2004

Nutritional genomics: the next frontier in the postgenomic era.

The interface between the nutritional environment and cellular/genetic processes is being referred to as "nutrigenomics." Nutrigenomics seeks to provide a molecular genetic understanding for how common dietary chemicals (i.e., nutrition) affect health by altering the expression and/or structure of an individual's genetic makeup. The fundamental concepts of the field are that the progression from a healthy phenotype to a chronic disease phenotype must occur by changes in gene expression or by differences in activities of proteins and enzymes and that dietary chemicals directly or indirectly regulate the expression of genomic information. We present a conceptual basis and specific examples for this new branch of genomic research that focuses on the tenets of nutritional genomics: 1) common dietary chemicals act on the human genome, either directly or indirectly, to alter gene expression or structure; 2) under certain circumstances and in some individuals, diet can be a serious risk factor for a number of diseases; 3) some diet-regulated genes (and their normal, common variants) are likely to play a role in the onset, incidence, progression, and/or severity of chronic diseases; 4) the degree to which diet influences the balance between healthy and disease states may depend on an individual's genetic makeup; and 5) dietary intervention based on knowledge of nutritional requirement, nutritional status, and genotype (i.e., "individualized nutrition") can be used to prevent, mitigate, or cure chronic disease.

Chronic Disease↗
PubMed2026

Rapid and exceptionally small-scale adaptation of the alpine plant Cardamine resedifolia to mining-contaminated soils in multi-stress condition.

The mechanisms by which plants tolerate soil contamination have been studied in details in controlled laboratory conditions, but they still remain largely unexplored in natural conditions where mixtures of contaminants are present in soils and their effects might interact with other environmental variables. This is especially true in high-altitude alpine environments, where abiotic stress is naturally heightened, but which so far have received little attention in environmental pollution studies. As we were interested in the tolerance mechanisms at play on very fine spatiotemporal scales for alpine plants growing under multi-stress conditions, we chose Cardamine resedifolia as our biological model. This plant is indeed frequently found in areas contaminated by Trace Metals and Metalloids and Polycyclic Aromatic Hydrocarbons in high elevation. We studied populations from former copper, silver-lead, and coal mines in alpine environments, along with populations growing on nearby reference soils. We measured genetic variability within populations as well as genetic differentiation between them, and tested for local adaptation to soil contamination using reciprocal transplants. Population pairs showing signs of local adaptation were then examined using genome scans to identify genes potentially under selection. We found high levels of genetic differentiation between populations growing on contaminated and reference soils a few dozen meters apart. In most cases local adaptation was detected, especially in former copper mines. Genome scans identified genes involved in metal stress management as potentially being under selection. This study provides evidence for rapid adaptation to human-induced pollution in alpine plants at remarkably small spatial scales. It offers new insights into the short-term ecological and evolutionary consequences of mining activities in alpine ecosystems, particularly in relation to substrate-driven differentiation.

Alpine plants↗
PubMed2001

Identification of a genomic island present in the majority of pathogenic isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a ubiquitous gram-negative bacterium, is capable of colonizing a wide range of environmental niches and can also cause serious infections in humans. In order to understand the genetic makeup of pathogenic P. aeruginosa strains, a method of differential hybridization of arrayed libraries of cloned DNA fragments was developed. An M13 library of DNA from strain X24509, isolated from a patient with a urinary tract infection, was screened using a DNA probe from P. aeruginosa strain PAO1. The genome of PAO1 has been recently sequenced and can be used as a reference for comparisons of genetic organization in different strains. M13 clones that did not react with a DNA probe from PAO1 carried X24509-specific inserts. When a similar array hybridization analysis with DNA probes from different strains was used, a set of M13 clones which carried sequences present in the majority of human P. aeruginosa isolates from a wide range of clinical sources was identified. The inserts of these clones were used to identify cosmids encompassing a contiguous 48.9-kb region of the X24509 chromosome called PAGI-1 (for "P. aeruginosa genomic island 1"). PAGI-1 is incorporated in the X24509 chromosome at a locus that shows a deletion of a 6,729-bp region present in strain PAO1. Survey of the incidence of PAGI-1 revealed that this island is present in 85% of the strains from clinical sources. Approximately half of the PAGI-1-carrying strains show the same deletion as X24509, while the remaining strains contain both the PAGI-1 sequences and the 6,729-bp PAO1 segment. Sequence analysis of PAGI-1 revealed that it contains 51 predicted open reading frames. Several of these genes encoded products with predictable function based on their sequence similarities to known genes, including insertion sequences, determinants of regulatory proteins, a number of dehydrogenase gene homologs, and two for proteins of implicated in detoxification of reactive oxygen species. It is very likely that PAGI-1 was acquired by a large number of P. aeruginosa isolates through horizontal gene transfer. The selection for its maintenance may be the consequence of expression of any one of the genes of unknown function or the genes which allow P. aeruginosa to survive under the conditions that generate reactive oxygen species. Alternatively, one or both of the transcriptional regulators encoded in PAGI-1 may control the expression of genes in the P. aeruginosa chromosome, which provides a selective advantage for strains that have acquired this genomic island.

Cloning, Molecular↗
PubMed2003

Evidence for quasispecies distributions in the human hepatitis A virus genome.

Nucleotide sequence analysis of multiple molecular clones of the hepatitis A virus (HAV), generated by reverse transcription-PCR of two capsid-coding regions, revealed a degree of heterogeneity compatible with a quasispecies structure in three clinical samples. Passage of plaque-purified reference strain HAV pHM175 43c in FRhK-4 cells documented the generation of a mutant distribution of HAV genomes. The mutant spectra showed mutation frequencies in the range of 1 x 10(-3) to 1 x 10(-4) substitutions per nucleotide, with a dominance of transition over transversion mutations. While in the VP3-coding region, nonsynonymous mutations were predominant; in the VP1-coding region they were uncommon. Around 50% of the amino acid replacements involved residues located at or near antigenic sites. Most of the detected mutations occurred at or in the vicinity of rare codons, suggesting a dynamics of mutation-selection, predominantly at and around rare codons. The results indicate that despite antigenic conservation, HAV replicates as a complex distribution of mutants, a feature of viral quasispecies.

Amino Acid Sequence↗
PubMed2005

Size of the protein-coding genome and rate of molecular evolution.

In diploid populations of size N, there will be 2 Nmu mutations per nucleotide (nt) site (or per locus) per generation (mu stands for mutation rate). If either the population or the coding genome double in size, one expects 4 Nmu mutations. What is important is not the population size per se but the number of genes (coding sites), the two being often interconverted. Here we compared the total physical length of protein-coding genomes (n) with the corresponding absolute rates of synonymous substitution (K(S)), an empirical neutral reference. In the classical occupancy problem and in the coupons collector (CC) problem, n was expressed as the mean rate of change (K(CC)). Despite inherently very low power of the approaches involving averaging of rates, the mode of molecular evolution of the total size phenotype of the coding genome could be evidenced through differences between the genomic estimates of K(CC) [K(CC)=1/(ln n + 0.57721) n] and rate of molecular evolution, K(S). We found that (1) the estimates of n and K(S) are reciprocally correlated across taxa (r=0.812; p<< 0.001); (2) the gamete-cell division hypothesis (Chang et al. Proc Natl Acad Sci USA 91:827-831, 1994) can be confirmed independently in terms of K(CC)/K(S) ratios; (3) the time scale of molecular evolution changes with change in mutation rate, as previously shown by Takahata (Proc Natl Acad Sci USA 87:2419-2423, 1990), Takahata et al. (Genetics 130:925-938, 1992), and Vekemans and Slatkin (Genetics 137:1157-1165, 1994); (4) the generation time and population size (Lynch and Conery, Science 302:1401-1404, 2003) effects left their "signatures" at the level of the size phenotype of the protein-coding genome.

Codon↗
PubMedDate not supplied

Genomic annotation and transcriptome analysis of the zebrafish (Danio rerio) hox complex with description of a novel member, hox b 13a.

The zebrafish (Danio rerio) is an important model in evolutionary developmental biology, and its study is being revolutionized by the zebrafish genome project. Sequencing is at an advanced stage, but annotation is largely the result of in silico analyses. We have performed genomic annotation, comparative genomics, and transcriptional analysis using microarrays of the hox homeobox-containing transcription factors. These genes have important roles in specifying the body plan. Candidate sequences were located in version Z v 4 of the Ensembl genome database by TBLASTN searching with Danio and other vertebrate published Hox protein sequences. Homologies were confirmed by alignment with reference sequences, and by the relative position of genes along each cluster. RT-PCR using adult Tübingen cDNA was used to confirm annotations, to check the genomic sequence and to confirm expression in vivo. Our RT-PCR and microarray data show that all 49 hox genes are expressed in adult zebrafish. Significant expression for all known hox genes could be detected in our microarray analysis. We also find significant expression of hox 8 paralogs and hox b 7 a in the anti-sense direction. A novel gene, D. rerio hox b 13 a, was identified, and a preliminary characterization by in situ hybridization showed expression at 24 hpf at the tip of the developing tail. We are currently characterizing this gene at the functional level. We argue that the oligo design for microarrays can be greatly enhanced by the availability of genomic sequences.

Animals↗
PubMed2003

Transcriptome profiling of a Saccharomyces cerevisiae mutant with a constitutively activated Ras/cAMP pathway.

Often changes in gene expression levels have been considered significant only when above/below some arbitrarily chosen threshold. We investigated the effect of applying a purely statistical approach to microarray analysis and demonstrated that small changes in gene expression have biological significance. Whole genome microarray analysis of a pde2Delta mutant, constructed in the Saccharomyces cerevisiae reference strain FY23, revealed altered expression of approximately 11% of protein encoding genes. The mutant, characterized by constitutive activation of the Ras/cAMP pathway, has increased sensitivity to stress, reduced ability to assimilate nonfermentable carbon sources, and some cell wall integrity defects. Applying the Munich Information Centre for Protein Sequences (MIPS) functional categories revealed increased expression of genes related to ribosome biogenesis and downregulation of genes in the cell rescue, defense, cell death and aging category, suggesting a decreased response to stress conditions. A reduced level of gene expression in the unfolded protein response pathway (UPR) was observed. Cell wall genes whose expression was affected by this mutation were also identified. Several of the cAMP-responsive orphan genes, upon further investigation, revealed cell wall functions; others had previously unidentified phenotypes assigned to them. This investigation provides a statistical global transcriptome analysis of the cellular response to constitutive activation of the Ras/cAMP pathway.

Cell Wall↗
PubMed2005

A genome-wide sequence-independent comparative analysis of insertion-deletion polymorphisms in multiple Mycobacterium tuberculosis strains.

We applied an enhanced version of subtractive hybridization for comparative analyses of indel differences between genomes of several Mycobacterium tuberculosis strains widespread in Russian regions, and the H37Rv reference strain. A number of differences were detected and partially analyzed, thus demonstrating the practicality of the approach. A majority of the insertions found were shared by all Russian strains, except for strain 1540 that revealed the highest virulence in animal tests. This strain possesses a number of genes absent from other clinical strains. Two of the differential genes were found to encode putative membrane proteins and are presumed to affect mycobacterial interaction with the host cell, thus enhancing virulent properties of the isolate. The method used is of general application, and enables the elaboration of a catalogue of indel polymorphic genomic differences between closely related strains.

Genetic Variation↗