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PubMed2005

Cultivating the uncultivated: a community genomics perspective.

Current isolation methods access only a small subset of the total microbial diversity. Although an isolate traditionally has been required for genomic characterization, the advent of sequencing of entire natural microbial communities enables culture-independent genomic analysis. Information about the genetic potential of uncultivated organisms can be used to predict the form of metabolic interdependencies and nutritional requirements. We believe that this could provide the information necessary to bypass bottlenecks that have inhibited cultivation of many microorganisms. However, it might not be practical or possible to isolate all of the vast number of microbial species and strains for laboratory-based characterization. Ultimately, cultivation-independent genomic and genomically enabled approaches could provide a way to directly analyze microbial activity in its geochemical and ecological context.

Bacteria↗

PubMed2001

Genomic structure and promoter characterization of an imprinted tumor suppressor gene ARHI.

We have recently identified a maternally imprinted tumor suppressor gene, ARHI (aplysia ras homolog I), the expression of which is lost in ovarian and breast cancers. We have now characterized the genomic structure of the gene including its promoter and the methylation status of its upstream CpG islands. The ARHI gene spans approximately 8 kb containing two exons and one intron. Exon 1 contains 81 non-translated nucleotides, connected to exon 2 with a 3.2-kb intron. The entire protein-coding region is located within exon 2 and encodes a 229-residue small GTP-binding protein belonging to the Ras superfamily. Genomic structure analysis has identified three potential CpG islands. Two of them (CpG island I and II) are located within the promoter and adjacent exon 1 of the ARHI gene. Aberrant methylation of these CpG islands has been detected in breast cancer cells but not in normal epithelial cells, supporting the possibility that appropriate methylation status of the CpG islands in the promoter region may play a role in the downregulation of ARHI gene expression. A TATA box is found 27 bp upstream of the transcription start site associated with several putative transcription factor binding sites. Transient transfection with nested deletion constructs of the 2-kb ARHI promoter regions fused to a luciferase reporter indicated a 121-bp sequence upstream of the transcription initiation site is required for basal promoter activity. Interestingly, this is the region where lower promoter activity has been observed in cancer cells than in normal cells.

Amino Acid Sequence↗

PubMed2002

Genomic structure and promoter characterization of the gene encoding the ErbB ligand betacellulin.

Betacellulin (BTC) belongs to the epidermal growth factor (EGF) family of peptide ligands that are characterized by a six-cysteine consensus motif (EGF-motif) that forms three intra-molecular disulfide bonds, crucial for binding the ErbB receptor family. A variety of in vitro studies have identified BTC as an important factor in the growth and/or differentiation of pancreatic islet cells. The molecular mechanisms that regulate the transcription of the BTC gene however have not been delineated. As an initial step, we have characterized the genomic structure of the mouse BTC (mBTC) gene. mBTC cDNA was used as a probe to screen a mouse 129/SVJ genomic bacterial artificial chromosome (BAC) library. Three positive clones containing the entire gene were isolated. DNA sequence analysis identified six exons (1-6) and five introns (A-E); a structure conserved among the EGF family. PCR analysis showed that introns A-E are approximately 7.8, 8.9, 3.8, 1.4 and 1.4 kb in length, respectively. The EGF-motif is encoded by exons 3 and 4 with an intron (intron C) disrupting the coding sequence between the second and third disulfide loops. All exon-intron boundaries are consistent with the "gt-ag" rule. Multiple transcription start sites and one poly(A) site, located 18 bp downstream of a polyadenylation signal sequence, were identified by 5'- and 3'-RACE, respectively. Approximately 2.6 kb of 5'-flanking region was sequenced and was shown to lack consensus TATA and CCAAT boxes, but was found to contain several putative cis-acting regulatory elements. These included consensus binding sites for transcription factors HNF3 beta, USF, Nkx2-5, AP-4, and Sp1. Functional promoter analysis of the 5'-flanking region in COS-7 cells, using 5'-deletion fragments (-168/+335; -635/+335; -732/+335; -1175/+335; -1698/+335) cloned into a promoterless firefly luciferase reporter vector, identified basal promoter activity and both positive and negative cis-acting elements.

5' Untranslated Regions↗

PubMed1981

Baboon endogenous virus genome: molecular cloning and structural characterization of nondefective viral genomes from DNA of a baboon cell strain.

Several heterogeneities in the baboon endogenous virus (BaEV) genomes that are present in the DNA of normal baboon tissues and the baboon cell strain BEF-3 have been described previously. To study these genomes, we cloned BaEV proviruses from BEF-3 cellular DNA into the lambda vector Charon 4A. Of the four full-length clones isolated, one was nondefective as determined by transfection. The sequence of a portion of this clone was found to code for amino acids 61-91 in the p30 region of the gag gene. This identification allowed us to align the restriction map with the BaEV genetic map. One heterogeneity, a BamHI site 2.4 kilobases (kb) from the proviral 5' end, was located close to the gag-pol junction; another, a BamHI site 1.4 kb from the 5' end of the genome, corresponded to the gag p30 coding sequence for amino acids 32-34; and a third, a Xho I site, was near the 3' end of the pol gene. To select the nondefective BaEV genomes from BEF-3 cells, we infected permissive cells with virus produced by BEF-3 cells and also transfected BEF-3 cellular DNA into permissive cells. The BaEV genomes in the permissive recipient cultures were then analyzed by restriction enzyme analysis. These nondefective genomes were found to be heterogeneous with respect to the gag-pol BamHI site and the Xho I site, but all were found to contain the BamHI site 1.4 kb from the 5' end of the genome.

Animals↗

PubMed2003

Genomic homogeneity between Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis belies their divergent growth rates.

BACKGROUND: Mycobacterium avium subspecies avium (M. avium) is frequently encountered in the environment, but also causes infections in animals and immunocompromised patients. In contrast, Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow-growing organism that is the causative agent of Johne's disease in cattle and chronic granulomatous infections in a variety of other ruminant hosts. Yet we show that despite their divergent phenotypes and the diseases they present, the genomes of M. avium and M. paratuberculosis share greater than 97% nucleotide identity over large (25 kb) genomic regions analyzed in this study. RESULTS: To characterize genome similarity between these two subspecies as well as attempt to understand their different growth rates, we designed oligonucleotide primers from M. avium sequence to amplify 15 minimally overlapping fragments of M. paratuberculosis genomic DNA encompassing the chromosomal origin of replication. These strategies resulted in the successful amplification and sequencing of a contiguous 11-kb fragment containing the putative Mycobacterium paratuberculosis origin of replication (oriC). This fragment contained 11 predicted open reading frames that showed a conserved gene order in the oriC locus when compared with several other Gram-positive bacteria. In addition, a GC skew analysis identified the origin of chromosomal replication which lies between the genes dnaA and dnaN. The presence of multiple DnaA boxes and the ATP-binding site in dnaA were also found in M. paratuberculosis. The strong nucleotide identity of M. avium and M. paratuberculosis in the region surrounding the origin of chromosomal replication led us to compare other areas of these genomes. A DNA homology matrix of 2 million nucleotides from each genome revealed strong synteny with only a few sequences present in one genome but absent in the other. Finally, the 16s rRNA gene from these two subspecies is 100% identical. CONCLUSIONS: We present for the first time, a description of the oriC region in M. paratuberculosis. In addition, genomic comparisons between these two mycobacterial subspecies suggest that differences in the oriC region may not be significant enough to account for the diverse bacterial replication rates. Finally, the few genetic differences present outside the origin of chromosomal replication in each genome may be responsible for the diverse growth rates or phenotypes observed between the avium and paratuberculosis subspecies.

Bacterial Proteins↗

PubMed2005

Genome-wide identification of the regulatory targets of a transcription factor using biochemical characterization and computational genomic analysis.

BACKGROUND: A major challenge in computational genomics is the development of methodologies that allow accurate genome-wide prediction of the regulatory targets of a transcription factor. We present a method for target identification that combines experimental characterization of binding requirements with computational genomic analysis. RESULTS: Our method identified potential target genes of the transcription factor Ndt80, a key transcriptional regulator involved in yeast sporulation, using the combined information of binding affinity, positional distribution, and conservation of the binding sites across multiple species. We have also developed a mathematical approach to compute the false positive rate and the total number of targets in the genome based on the multiple selection criteria. CONCLUSION: We have shown that combining biochemical characterization and computational genomic analysis leads to accurate identification of the genome-wide targets of a transcription factor. The method can be extended to other transcription factors and can complement other genomic approaches to transcriptional regulation.

Amino Acid Motifs↗

PubMed2005

Construction and characterization of a genomic BAC library for the Mus m. musculus mouse subspecies (PWD/Ph inbred strain).

BACKGROUND: The genome of classical laboratory strains of mice is an artificial mosaic of genomes originated from several mouse subspecies with predominant representation (>90%) of the Mus m. domesticus component. Mice of another subspecies, East European/Asian Mus m. musculus, can interbreed with the classical laboratory strains to generate hybrids with unprecedented phenotypic and genotypic variations. To study these variations in depth we prepared the first genomic large insert BAC library from an inbred strain derived purely from the Mus m. musculus-subspecies. The library will be used to seek and characterize genomic sequences controlling specific monogenic and polygenic complex traits, including modifiers of dominant and recessive mutations. RESULTS: A representative mouse genomic BAC library was derived from a female mouse of the PWD/Ph inbred strain of Mus m. musculus subspecies. The library consists of 144,768 primary clones from which 97% contain an insert of 120 kb average size. The library represents an equivalent of 6.7 x mouse haploid genome, as estimated from the total number of clones carrying genomic DNA inserts and from the average insert size. The clones were arrayed in duplicates onto eight high-density membranes that were screened with seven single-copy gene probes. The individual probes identified four to eleven positive clones, corresponding to 6.9-fold coverage of the mouse genome. Eighty-seven BAC-ends of PWD/Ph clones were sequenced, edited, and aligned with mouse C57BL/6J (B6) genome. Seventy-three BAC-ends displayed unique hits on B6 genome and their alignment revealed 0.92 single nucleotide polymorphisms (SNPs) per 100 bp. Insertions and deletions represented 0.3% of the BAC end sequences. CONCLUSION: Analysis of the novel genomic library for the PWD/Ph inbred strain demonstrated coverage of almost seven mouse genome equivalents and a capability to recover clones for specific regions of PWD/Ph genome. The single nucleotide polymorphism between the strains PWD/Ph and C57BL/6J was 0.92/100 bp, a value significantly higher than between classical laboratory strains. The library will serve as a resource for dissecting the phenotypic and genotypic variations between mice of the Mus m. musculus subspecies and classical laboratory mouse strains.

Animals↗

PubMed2002

Bioinformatics, functional genomics, and proteomics study of Bacillus sp.

The ability of bioinformatics to characterize genomic and proteomic sequences from bacteria Bacillus sp. for prediction of genes and proteins has been evaluated. Genomics coupling with proteomics, which is relied on integration of the significant advances recently achieved in two-dimensional (2-D) electrophoretic separation of proteins and mass spectrometry (MS), are now important and high throughput techniques for qualifying and analyzing gene and protein expression, discovering new gene or protein products, and understanding of gene and protein functions including post-genomic study. In addition, the bioinformatics of Bacillus sp. is embraced into many databases that will facilitate to rapidly search the information of Bacillus sp. in both genomics and proteomics. It is also possible to highlight sites for post-translational modifications based on the specific protein sequence motifs that play important roles in the structure, activity and compartmentalization of proteins. Moreover, the secreted proteins from Bacillus sp. are interesting and widely used in many applications especially biomedical applications that are the highly advantages for their potential therapeutic values.

Bacillus↗

PubMed2005

Identification of a novel homozygous deletion region at 6q23.1 in medulloblastomas using high-resolution array comparative genomic hybridization analysis.

PURPOSE: The aim of this study is to comprehensively characterize genome copy number aberrations in medulloblastomas using high-resolution array comparative genomic hybridization. EXPERIMENTAL DESIGN: High-density genomic arrays containing 1,803 BAC clones were used to define recurrent chromosomal regions of gains or losses throughout the whole genome of medulloblastoma. A series of 3 medulloblastoma cell lines and 16 primary tumors were investigated. RESULTS: The detected consistent chromosomal aberrations included gains of 1q21.3-q23.1 (36.8%), 1q32.1 (47.4%), 2p23.1-p25.3 (52.6%), 7 (57.9%), 9q34.13-q34.3 (47.4%), 17p11.2-q25.3 (89.5%), and 20q13.31-q13.33 (42.1%), as well as losses of 3q26.1 (57.9%), 4q31.23-q32.3 (42.1%), 6q23.1-25.3 (57.9%), 8p22-23.3 (79%), 10q24.32-26.2 (57.9%), and 16q23.2-q24.3 (63.2%). One of the most notable aberrations was a homozygous deletion on chromosome 6q23 in the cell line DAOY, and single copy loss on 30.3% primary tumors. Further analyses defined a 0.887 Mbp minimal region of homozygous deletion at 6q23.1 flanked by markers SHGC-14149 (6q22.33) and SHGC-110551 (6q23.1). Quantitative reverse transcription-PCR analysis showed complete loss of expression of two genes located at 6q23.1, AK091351 (hypothetical protein FLJ34032) and KIAA1913, in the cell line DAOY. mRNA levels of these genes was reduced in cell lines D283 and D384, and in 50% and 70% of primary tumors, respectively. CONCLUSION: Current array comparative genomic hybridization analysis generates a comprehensive pattern of chromosomal aberrations in medulloblastomas. This information will lead to a better understanding of medulloblastoma tumorigenesis. The delineated regions of gains or losses will indicate locations of medulloblastoma-associated genes. A 0.887 Mbp homozygous deletion region was newly identified at 6q23.1. Frequent detection of reduced expression of AK091351 and KIAA1913 genes implicates them as suppressors of medulloblastoma tumorigenesis.

Adolescent↗

PubMed2003

Cloning and characterization of a second alpha-amylase gene (LKA2) from Lipomyces kononenkoae IGC4052B and its expression in Saccharomyces cerevisiae.

Lipomyces kononenkoae secretes a battery of highly effective amylases (i.e. alpha-amylase, glucoamylase, isoamylase and cyclomaltodextrin glucanotransferase activities) and is therefore considered as one of the most efficient raw starch-degrading yeasts known. Previously, we have cloned and characterized genomic and cDNA copies of the LKA1 alpha-amylase gene from L. kononenkoae IGC4052B (CBS5608T) and expressed them in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Here we report on the cloning and characterization of the genomic and cDNA copies of a second alpha-amylase gene (LKA2) from the same strain of L. kononenkoae. LKA2 was cloned initially as a 1663 bp cDNA harbouring an open reading frame (ORF) of 1496 nucleotides. Sequence analysis of LKA2 revealed that this ORF encodes a protein (Lka2p) of 499 amino acids, with a predicted molecular weight of 55,307 Da. The LKA2-encoded alpha-amylase showed significant homology to several bacterial cyclomaltodextrin glucanotransferases and also to the alpha-amylases of Aspergillus nidulans, Debaryomyces occidentalis, Saccharomycopsis fibuligera and Sz. pombe. When LKA2 was expressed under the control of the phosphoglycerate kinase gene promoter (PGK1(p)) in S. cerevisiae, it was found that the genomic copy contained a 55 bp intron that impaired the production of biologically active Lka2p in the heterologous host. In contrast to the genomic copy, the expression of the cDNA construct of PGK1p-LKA2 in S. cerevisiae resulted in the production of biologically active alpha-amylase. The LKA2-encoded alpha-amylase produced by S. cerevisiae exhibited a high specificity towards substrates containing alpha-1,4 glucosidic linkages. The optimum pH of Lka2p was found to be 3.5 and the optimum temperature was 60 degrees C. Besides LKA1, LKA2 is only the second L. kononenkoae gene ever cloned and expressed in S. cerevisiae. The cloning, characterization and co-expression of these two genes encoding these highly efficient alpha-amylases form an important part of an extensive research programme aimed at the development of amylolytic strains of S. cerevisiae for the efficient bioconversion of starch into commercially important commodities.

Amino Acid Sequence↗

PubMed1990

Genetics of human breast cancer.

During the past five years, important progress has been made in the identification of genes and/or chromosomal rearrangements that may have direct relevance for the development of human breast cancer. Along with rapid advances in the field of human genome characterization, the number of molecular tools to examine the genomic changes occurring in breast cancer cells is expanding rapidly. Therefore, the major challenge is not so much the identification of tumour specific genome alterations, but rather the interpretation of the findings in terms of biological implications. Since clearly defined precursor lesions are lacking in breast cancer, a multidisciplinary approach will be necessary to arrive at a more complete understanding of the established genetic alterations.

Breast Neoplasms↗

PubMed2025

Validation of caprine H11 and the Rosa26 platform for transgene integration via CRISPR-based system: investigations on stable transgene expression and genetic biosafety.

CRISPR/Cas9 technology is an efficient tool for site-specific livestock gene editing. However, to minimize potential disruption of host genome function, exogenous genes should be integrated into well-characterized genomic loci, such as H11 or Rosa26, which have been empirically validated for stable transgene expression. This study established a multi-dimensional assessment system to evaluate the biological applicability of the H11 locus and the widely used Rosa26 targeting platform as sites for targeted integration of exogenous genes in goats. Donor cells carrying the enhanced green fluorescent protein (EGFP) reporter gene at the H11 and Rosa26 loci were generated via CRISPR/Cas9-mediated homology-directed repair; this was followed by somatic cell nuclear transfer to produce transgenic cloned embryos and healthy offspring. Multi-dimensional analyses revealed the following. At the cellular level, there was stable and efficient EGFP expression at integration sites, with donor cells maintaining normal cell cycle progression, proliferation capacity, and apoptosis levels, and with no alterations in the transcriptional integrity of adjacent genes. At the embryonic level, there was sustained EGFP expression across pre-implantation embryonic stages, with developmental metrics statistically indistinguishable from wild-type embryos. Finally, at the individual level, cloned offspring exhibited growth phenotypes consistent with wild-type counterparts, and EGFP showed broad-spectrum expression in eight tissues. This study establishes the first CRISPR/Cas9-based crossscale (cellular-embryonic-individual) validation in goats, demonstrating that the H11 and Rosa26 loci support efficient and stable transgene integration in goats. These results provide a precise and predictable technical framework for livestock genetic improvement.

Animals↗

PubMed2026

The genetic control of rapid genome content divergence in Arabidopsis thaliana.

Genome evolution in eukaryotes is predominantly driven by the dynamics of repetitive sequences, which vary widely in both copy number and sequence composition. Rates of repeat evolution differ between and within species and are likely modulated by both genetics and environment. To uncover factors shaping the rate of genome content evolution, we analyzed 1,142 resequenced Arabidopsis thaliana genomes using a novel K-mer based approach to characterize genome content variation and identify hypervariable regions underlying differences in repeat abundance. We next treated repeat abundance as a quantitative trait and performed genome-wide association analyses across more than 400 repeat families to identify the genetic basis of copy number variation. Integrating these results through a meta-GWAS approach revealed both cis-acting variants and more than 50 trans-acting loci that regulate repeat abundance genome-wide. Cis-acting variation was predominantly localized to pericentromeric and centromeric regions, whereas trans-acting loci were enriched for candidate genes involved in DNA replication, DNA repair, DNA methylation regulation. Finally, we found evidence that purifying selection acts against mutations that accelerate genome content divergence, favoring alleles that constrain repeat expansion. Together, these findings provide new insights into the genetic architecture and evolutionary forces shaping genome evolution in A. thaliana and establish a framework for investigating these processes in other plant species.

Journal Article↗

PubMed2006

Distribution of microsatellites in the genome of Medicago truncatula: a resource of genetic markers that integrate genetic and physical maps.

Microsatellites are tandemly repeated short DNA sequences that are favored as molecular-genetic markers due to their high polymorphism index. Plant genomes characterized to date exhibit taxon-specific differences in frequency, genomic location, and motif structure of microsatellites, indicating that extant microsatellites originated recently and turn over quickly. With the goal of using microsatellite markers to integrate the physical and genetic maps of Medicago truncatula, we surveyed the frequency and distribution of perfect microsatellites in 77 Mbp of gene-rich BAC sequences, 27 Mbp of nonredundant transcript sequences, 20 Mbp of random whole genome shotgun sequences, and 49 Mbp of BAC-end sequences. Microsatellites are predominantly located in gene-rich regions of the genome, with a density of one long (i.e., > or = 20 nt) microsatellite every 12 kbp, while the frequency of individual motifs varied according to the genome fraction under analysis. A total of 1,236 microsatellites were analyzed for polymorphism between parents of our reference intraspecific mapping population, revealing that motifs (AT)n, (AG)n, (AC)n, and (AAT)n exhibit the highest allelic diversity. A total of 378 genetic markers could be integrated with sequenced BAC clones, anchoring 274 physical contigs that represent 174 Mbp of the genome and composing an estimated 70% of the euchromatic gene space.

Alleles↗

PubMed2026

The genetic control of rapid genome content divergence in Arabidopsis thaliana.

Genome evolution in eukaryotes is predominantly driven by the dynamics of repetitive sequences, which vary widely in both copy number and sequence composition. Rates of repeat evolution differ between and within species and are likely modulated by both genetics and environment. To uncover factors shaping the rate of genome content evolution, we analyzed 1043 resequenced Arabidopsis thaliana genomes using a novel K-mer-based approach to characterize genome content variation and identify hypervariable regions underlying differences in repeat abundance. We next treated repeat abundance as a quantitative trait and performed genome-wide association analyses across more than 400 repeat families to identify the genetic basis of copy number variation. Integrating these results through a meta-GWAS approach revealed both cis-acting variants and more than 50 candidate trans-acting loci associated with repeat abundance genome-wide. Cis-acting variation was predominantly localized to pericentromeric and centromeric regions, whereas trans-acting loci were enriched for candidate genes involved in DNA replication, DNA repair, and DNA methylation regulation. The results are consistent with purifying selection acting against mutations that accelerate genome content divergence, favoring alleles that constrain repeat expansion. Together, these findings provide new insights into the genetic architecture and evolutionary forces shaping genome evolution in A. thaliana and establish a framework for investigating these processes in other plant species.

Arabidopsis↗

PubMed2003

Molecular marker dissection of rice (Oryza sativa L.) plant architecture under temperate and tropical climates.

Rice ( Oryza sativa L.) plants develop vertically with shoot elongation and horizontally with tillering. The purpose of this study was to identify and characterize genomic regions influencing the rice plant architecture by quantitative trait locus (QTL) analysis for the component traits: culm length (CL), panicle length (PnL), panicle number (PnN) and tiller number (TN). For this QTL analysis, 191 recombinant inbred lines (F(7)) derived from a cross of Milyang 23 (M23) and Akihikari (AK) were grown in 1995, 1996 and 1997 (May-Oct) in Joetsu, Japan (temperate climate), and in the 2000 dry season (Jan-Apr), the 2000 wet season (Jun-Oct) and the 2001 dry season in Los Baños, The Philippines (tropical climate). Results showed that rice plant architecture was influenced by 19 genomic regions categorized into five groups. In Group I, two regions (on chrs. 6 and 11) affected shoot elongation (CL and PnL) and tillering (PnN and TN) in opposite directions more significantly in Los Baños than in Joetsu. In Group II, two regions (chrs. 3 and 12) affected shoot elongation, whereas in Group III, five regions [chrs. 1 (two), 2, 3 and 9] affected only culm length (CL). Expressions of four regions of Group III were influenced by either tropical or temperate environments. In Group IV, seven regions (chrs. 1, 2, 4, 5, 6, 8 and 9) controlled panicle development (PnN or PnL), and in Group V, three regions (chrs. 1, 2 and 3) regulated tillering (PnN or TN). Characterizing these 19 genomic regions provided a detailed analysis of rice plant architecture with emphasis on the multiple effect and environmental responsive regions.

Genetic Markers↗

PubMed2026

Longitudinal whole-genome analysis of bluetongue virus identifies conserved serotype-specific genomes and distinct genomic constellations within a Colorado sheep flock (2021-2023).

Bluetongue virus (BTV) is a segmented double-stranded RNA virus of ruminants transmitted by Culicoides spp. biting midges. Although the genome consists of ten segments, classification into serotypes is primarily based on genome segment 2. However, reassortment among genomic segments is a major driver of BTV evolution and diversity. This study used longitudinal whole-genome sequencing to characterize BTV genomes collected from 2021 to 2023 within a single sheep flock in Colorado, where multiple serotypes co-circulate. Whole-genome sequences were generated from fourteen blood samples representing four serotypes: BTV-6, -11, -13, and -17. Longitudinal sampling identified multiple BTV serotypes within individual sheep across consecutive years. Tanglegram analysis comparing segment phylogenies to the segment 2 tree demonstrated incongruent topologies across all genomic segments, suggestive of reassortment or the circulation of distinct genomic constellations. Nucleotide-level comparisons revealed high sequence homology among same-serotype samples from the same year, while the greatest genetic divergence was observed among BTV-17 genomes collected in different years. Additionally, all BTV-13 genomes contained a previously undescribed nonsynonymous substitution in segment 10 predicted to extend the encoded protein by three amino acids. Together, these findings demonstrate that highly conserved BTV genomes and distinct genomic constellations can be detected at the flock level across multiple years. This longitudinal whole-genome approach reveals the genetic complexity of endemic BTV populations, including novel variants and genomic patterns consistent with reassortment that are lost with conventional serotyped-based approaches, highlighting the need to integrate whole-genome characterization into endemic BTV monitoring programs.

Animals↗

PubMed1995

Chromosomal location of endogenous geminivirus-related DNA sequences in Nicotiana tabacum L.

The N. tabacum (tobacco) nuclear genome carries approximately 25 multiple direct repeats of a geminivirus-related DNA (GRD) sequence that probably arose by illegitimate recombination, following geminivirus infection, during Nicotiana evolution. Each GRD repeat carries sequences similar to the geminiviral AL1 gene of the tomato golden mosaic virus (TGMV), encoding a protein required for viral DNA replication, plus the cis-essential replication origin. Using a cloned 14-kb GRD repeat sequence as a probe for fluorescence in situ hybridization (FISH), we identified a unique tobacco chromosome carrying GRD. Translocations between chromosomes of the tobacco S and T genomes were used as physical markers by sequentially hybridizing chromosomes with labelled GRD and total genomic DNA from N. sylvestris (equivalent to the S genome). The 25S, 18S and 5.8S ribosomal gene clusters were detected in double-labelling experiments for use as additional markers to identify the chromosomal location of GRD. GRD occupies one site on a homologous pair of small submetacentrics from the T genome characterized by a lack of either translocated segments from the S genome or ribosomal genes. GRD provides an additional marker for the small chromosomes of the T genome and a useful phylogenetic tool.

Chromosome Mapping↗