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Porcine adenoviruses: an update on genome analysis and vector development.

Although porcine adenoviruses (PAdV) are present in the swine populations worldwide, they usually do not cause any disease, or the infection is only manifested in a mild diarrhoea or respiratory signs. The importance of adenoviruses, however, is constantly growing as there is a possibility of developing them into viral vector vaccines against more significant swine pathogens. A short summary of the well-established facts of porcine adenoviruses is given and recent developments of the genetic analysis of these viruses are discussed in detail. The possibilities of vector development and examples of vector vaccines already reported in the literature are mentioned.

Animals↗

A comparative genomic analysis of left- and right-sided colon cancer using real-world data from the AACR project GENIE BPC dataset.

Left- and Right-sided colon cancers (LCC and RCC) are increasingly recognized as distinct clinicopathological and molecular subtypes with divergent prognoses and therapeutic responses. Leveraging a large, multi-institutional cohort from the AACR Project Genomics Evidence Neoplasia Information Exchange (GENIE) Biopharma Collaborative (BPC) (n = 750; LCC: 363 vs. RCC: 387), we conducted a comprehensive analysis of mutational profiles, tumor mutation burden (TMB), and survival outcomes. Our findings revealed a markedly higher TMB in RCC compared to LCC (6.65 &#xb1; 11.3 vs. 3.17 &#xb1; 4.35; adjusted P = 3.12&#xd7;10-32), suggesting greater genomic instability in RCC. After applying functional annotation filters (PolyPhen > 0.85, SIFT < 0.05), RCC tumors were significantly enriched for mutations in BRAF (23.1% vs. 6.7%), KMT2D (8.6% vs. 3.2%), and SMAD4 (13.1% vs. 7.3%), while TP53 mutations predominated in LCC (40.6% vs. 31.8%). Multivariate Cox regression analysis identified RCC as an independent predictor of poorer overall survival (OS) relative to LCC (HR: 1.30, 95% CI: 1.02-1.66, P = 0.033). Notably, KRAS mutations were associated with significantly worse OS in LCC (HR: 1.68, 95% CI: 1.06-2.70, P = 0.027), while BRAF mutations predicted adverse outcomes in RCC (HR: 1.58, 95% CI: 1.05-2.37, P = 0.028). These results underscore the prognostic value of tumor sidedness and specific genetic alterations in colon adenocarcinoma. Our study highlights the need for sidedness-specific molecular profiling to inform precision oncology strategies in colon cancer management.

BRAF↗

Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial.

BACKGROUND: Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important opportunistic human pathogen. Generally, the acquisition of genes in the form of pathogenicity islands distinguishes pathogenic isolates from nonpathogens. We therefore sequenced a highly virulent strain of P. aeruginosa, PA14, and compared it with a previously sequenced (and less pathogenic) strain, PAO1, to identify novel virulence genes. RESULTS: The PA14 and PAO1 genomes are remarkably similar, although PA14 has a slightly larger genome (6.5 megabses [Mb]) than does PAO1 (6.3 Mb). We identified 58 PA14 gene clusters that are absent in PAO1 to determine which of these genes, if any, contribute to its enhanced virulence in a Caenorhabditis elegans pathogenicity model. First, we tested 18 additional diverse strains in the C. elegans model and observed a wide range of pathogenic potential; however, genotyping these strains using a custom microarray showed that the presence of PA14 genes that are absent in PAO1 did not correlate with the virulence of these strains. Second, we utilized a full-genome nonredundant mutant library of PA14 to identify five genes (absent in PAO1) required for C. elegans killing. Surprisingly, although these five genes are present in many other P. aeruginosa strains, they do not correlate with virulence in C. elegans. CONCLUSION: Genes required for pathogenicity in one strain of P. aeruginosa are neither required for nor predictive of virulence in other strains. We therefore propose that virulence in this organism is both multifactorial and combinatorial, the result of a pool of pathogenicity-related genes that interact in various combinations in different genetic backgrounds.

Base Sequence↗

MAAP: a versatile and universal tool for genome analysis.

Multiple arbitrary amplicon profiling (MAAP) uses one or more oligonucleotide primers (> or = 5 nt) of arbitrary sequence to initiate DNA amplification and generate characteristic fingerprints from anonymous genomes or DNA templates. MAAP markers can be used in general fingerprinting as well as in mapping applications, either directly or as sequence-characterized amplified regions (SCARs). MAAP profiles can be tailored in the number of monomorphic and/or polymorphic products. For example, multiple endonuclease digestion of template DNA or the use of mini-hairpin primers can enhance detection of polymorphic DNA. Comparison of the expected and actual number of amplification products produced with primers differing in length, sequence and GC content from templates of varying complexity reveal severe departures from theoretical formulations with interesting implications in primer-template interaction. Extensive primer-template mismatching can occur when using templates of low complexity or long primers. Primer annealing and extension appears directed by an 8 nt 3'-terminal primer domain, requires sites with perfect homology to the first 5-6 nt fom the 3' terminus, and involves direct physical interaction between amplicon annealing sites.

Animals↗

Genomic analysis of blue nevi and related dermal melanocytic proliferations.

Blue nevi are benign dermal melanocytic proliferations that can sometimes share overlapping microscopic features with melanoma. We used comparative genomic hybridization to analyze three groups of dermal melanocytic proliferations. Group 1 consisted of 10 cellular blue nevi and 1 deep penetrating nevus, none of which showed chromosomal aberrations. Group 2 consisted of 11 lesions that were histopathologically ambiguous. Three of these lesions demonstrated chromosomal aberrations (three or fewer per lesion). Group 3 consisted of seven histopathologically malignant lesions, each showing three or more chromosomal aberrations. Moderate to severe cytologic atypia and a mitotic rate of three or more mitoses per 10 high power fields were present in six of eight (75%) lesions that had at least three chromosomal aberrations but were absent in 15 of 20 (75%) lesions without chromosomal aberrations. Necrosis was present in four of the 29 (13%) lesions, with every lesion with necrosis demonstrating three or more genomic abnormalities. In conclusion, histopathologically unequivocally benign or malignant dermal melanocytic proliferations show nonoverlapping patterns of chromosomal aberrations. Ambiguous lesions can be separated into lesions with and without chromosomal aberrations. Future studies with clinical follow-up are necessary to determine which aberrations are most informative for classification of these lesions.

Adolescent↗

Genomic analysis of biochemical function.

Establishing the linkage between an individual biochemical activity and the gene(s) specifying that activity has been facilitated by advances in mass spectrometry and affinity purification methods. In addition, a genomic protein array has been produced in yeast by fusing each yeast open reading frame to glutathione-S-transferase, thus linking each protein with its cognate gene. Purification and biochemical assay of pools of glutathione-S-transferase-open-reading-frame proteins allows analysis of the entire proteome for biochemical activities, followed by simple deconvolution to identify the responsible open reading frame. An alternative method to analyze large sets of proteins is the use of protein microarrays in which over 10,000 individual proteins can be immobilized and assayed on a single slide.

Animals↗

The protein kinase ERK3 is encoded by a single functional gene: genomic analysis of the ERK3 gene family.

Extracellular signal-regulated kinase 3 (ERK3) is a distantly related member of the mitogen-activated protein (MAP) kinase family of serine/threonine kinases. Here, we report the characterization of the genomic loci encoding ERK3 in mice and humans. The mouse ERK3 gene (Mapk6) spans more than 20 kb and is split into six exons. Its structure is similar to that of the human MAPK6 gene, which extends over 40 kb. We also identified and characterized a mouse Mapk6 processed pseudogene. In humans, database analysis has revealed the presence of six MAPK6 processed pseudogenes localized on four different chromosomes. We further show that the structure of MAPK6 is closely related to that of the gene encoding the homologous protein kinase p63(MAPK) (MAPK4), suggesting that the two genes arose by duplication. Our analysis demonstrates that the ERK3 subfamily of MAP kinase genes is composed of two functional genes, MAPK6 and MAPK4, and several pseudogenes.

Animals↗

Expression of the vaccinia virus genome: analysis and mapping of mRNAs encoded within the inverted terminal repetition.

We have investigated the organization of transcriptional units within a 9000 bp segment of the terminally repeated region of the DNA genome of vaccinia virus, which uses its own enzyme system to synthesize mRNA within the cytoplasm of infected cells. RNA splicing, which has been demonstrated for DNA viruses that replicate within the nucleus of infected cells, does not appear to be involved in the formation of these first vaccinia virus mRNAs to be examined. Three immediate early mRNAs, approximately 1050, 600 and 1100 nucleotides long, were mapped between 3.21 and 4.24, 6.54 and 7.16, and 7.20 and 8.23 kb from the end of the genome, respectively. The direction of transcription was toward the end of the genome for the two larger mRNAs and in the opposite direction for the smallest one. Additional minor RNAs, which were larger in size, were mapped between and to the same DNA strand as the mRNAs of 1050 and 1100 nucleotides. No evidence for interrupted genes was obtained by nuclease S1 analysis after hybridization of RNA to labeled DNA. In addition, the 5' ends of the mRNAs, which were specifically labeled by in vitro capping, hybridized to DNA adjacent to the body of the message.

DNA, Recombinant↗

Integrative transcriptomic, spatial and functional-genomic analysis identifies a UFMylation-related vascular-stromal program and prioritizes WWTR1 in glioblastoma.

Glioblastoma (GBM) contains spatially organized stress-adaptive and vascular niches. Because transcript abundance does not measure UFM1 conjugation, we asked whether a UFMylation-related transcriptional axis identifies a reproducible tissue program and alters candidate prioritization. In 518 unique primary TCGA-GBM tumors profiled on the Affymetrix HT Human Genome U133A array, weighted gene co-expression network analysis of 8,000 variable genes yielded 12 modules. The 278-gene green module ranked first across nine prespecified traits (mean |r|=0.637). Direct overlap comprised 1/3 measurable UFMylation-core, 5/19 ER-stress/UPR, and 2/15 proteostasis genes; after excluding overlapping genes, correlations with the green eigengene remained significant (r&#x2009;=&#x2009;0.373, 0.831, 0.639, and 0.699 for UFMylation-core, ER-stress/UPR, proteostasis, and composite scores, respectively). The green score was associated with overall survival per standard-deviation increase (HR 1.17, 95% CI 1.07-1.28), although clinical adjustment attenuated the estimate. In a 10-sample single-cell dataset, sample-level scores were higher in pericytes and endothelial cells than in malignant cells. Donor-aware IvyGAP analysis supported regional organization, whereas one Visium section showed stronger concordance with ER-stress/UPR and mesenchymal scores than with the UFMylation-core score. CellChat indicated pathway-selective rather than global remodeling of inferred vascular communication. Layer ablation moved WWTR1 from rank 48 using WGCNA alone to rank 4 overall and rank 1 among non-common-essential genes after cross-platform integration. These findings define an ER-stress/mesenchymal-weighted, UFMylation-related vascular-stromal transcriptional association and nominate WWTR1 for experimental testing.

Humans↗

Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus.

The lactic acid bacterium Streptococcus thermophilus is widely used for the manufacture of yogurt and cheese. This dairy species of major economic importance is phylogenetically close to pathogenic streptococci, raising the possibility that it has a potential for virulence. Here we report the genome sequences of two yogurt strains of S. thermophilus. We found a striking level of gene decay (10% pseudogenes) in both microorganisms. Many genes involved in carbon utilization are nonfunctional, in line with the paucity of carbon sources in milk. Notably, most streptococcal virulence-related genes that are not involved in basic cellular processes are either inactivated or absent in the dairy streptococcus. Adaptation to the constant milk environment appears to have resulted in the stabilization of the genome structure. We conclude that S. thermophilus has evolved mainly through loss-of-function events that remarkably mirror the environment of the dairy niche resulting in a severely diminished pathogenic potential.

Bacterial Proteins↗

Complete genome analysis and molecular characterization of Usutu virus that emerged in Austria in 2001: comparison with the South African strain SAAR-1776 and other flaviviruses.

Here we describe the complete genome sequences of two strains of Usutu virus (USUV), a mosquito-borne member of the genus Flavivirus in the Japanese encephalitis virus (JEV) serogroup. USUV was detected in Austria in 2001 causing a high mortality rate in blackbirds; the reference strain (SAAR-1776) was isolated in 1958 from mosquitoes in South Africa and has never been associated with avian mortality. The Austrian and South African isolates exhibited 97% nucleotide and 99% amino acid identity. Phylogenetic trees were constructed displaying the genetic relationships of USUV with other members of the genus Flavivirus. When comparing USUV with other JEV serogroup viruses, the closest lineage was Murray Valley encephalitis virus (nt: 73%, aa: 82%) followed by JEV (nt: 71%, aa: 81%) and West Nile virus (nt: 68%, aa: 75%). Comparison of the genomes showed that the conserved structural elements and putative enzyme motifs were homologous in the two USUV strains and the JEV serogroup. The factors that determine the severe clinical symptoms caused by the Austrian USUV strain in Eurasian blackbirds are discussed. We also offer a possible explanation for the origins and dispersal of USUV, JEV, and MVEV out of Africa.

Amino Acid Sequence↗

Genomic analysis of cultivated barley (Hordeum vulgare) using sequence-tagged molecular markers. Estimates of divergence based on RFLP and PCR markers derived from stress-responsive genes, and simple-sequence repeats (SSRs).

Three types of molecular markers have been compared for their utility in evaluating genetic diversity among cultivars of Hordeum vulgare. Restriction fragment length polymorphisms at 71 sites were scored with the aid of probes corresponding to stress-responsive genes from barley and wheat, coding for a low-molecular-weight heat shock protein, a dehydrin, an aldose reductase homolog, and a 18.9-kDa drought-induced protein of unknown function. Indexes of genetic diversity computed in the total sample and within groups of cultivars (two-rowed and six-rowed, winter and spring varieties) indicated high values of genetic differentiation ( F (ST) >15%). A second assessment of genetic diversity was performed by PCR amplification of genomic DNA using as primers 13 arbitrary oligonucleotides derived from sequences of the same stress-responsive genes. A high degree of polymorphism was uncovered using these markers also, but they yielded low values for F (ST) (<7%) among groups of cultivars. Finally, 15 different simple-sequence repeats (AC or AG) were amplified with primers based on unique flanking sequences. Levels of polymorphism and differentiation between groups of cultivars revealed by these markers were quite high. Ordination techniques applied to measures of genetic distance among cultivars demonstrated a remarkable ability of the RFLPs associated with stress-responsive genes to discriminate on the basis of growth habit. The correlation with production data for the cultivars in different environments was also significant. This "functional genomics" strategy was therefore as informative as the "structural genomics" (SSR-based) approach, but requires the analysis of fewer probes.

Base Sequence↗

Comparative genomic analysis of Artemisia argyi reveals asymmetric expansion of terpene synthases and conservation of artemisinin biosynthesis.

Artemisia argyi, a perennial herb of the Asteraceae family, possesses significant therapeutic and economic value. We present a 7.88&#x2009;Gb chromosome-level haplotype-resolved genome assembly, revealing its unique evolutionary trajectory. The karyotype (2n&#x2009;=&#x2009;34) of A. argyi is that of an autotetraploid, which underwent gametic chromosome fusion prior to species-specific whole-genome duplication (WGD-3). The genome exhibits pronounced multivalent chromosome pairing and frequent recombination among homologous groups. Asymmetrical evolution following WGD-3 is a hallmark feature, evidenced by imbalanced allelic gene loss and widespread neofunctionalization. The terpene synthase (TPS) gene family exemplifies this pattern, having expanded through four duplication events in A. argyi. Recent tandem duplications and allelic functional differentiation have generated substantial gene functional diversity. Notably, we identified a tandem-duplicated six-copy ADS homolog (AarADS)-a key TPS gene in the artemisinin biosynthetic pathway of Artemisia annua (AanADS)-localized exclusively to a single chromosome in A. argyi. Unlike AanADS, which converts farnesyl pyrophosphate (FPP) to amorpha-4,11-diene, AarADS catalyzes FPP to &#x3b1;-bisabolol. Evolutionary analysis suggested that AanADS acquired its specialized function via a derived mutation in the A. annua lineage. This study elucidates the genomic evolution underpinning A. argyi's distinctive medicinal properties.

Alkyl and Aryl Transferases↗

High levels of RNA editing in a vascular plant chloroplast genome: analysis of transcripts from the fern Adiantum capillus-veneris.

We sequenced transcripts from all putative genes for proteins, rRNAs, and a selection of gene-encoding tRNAs in the chloroplast genome of the fern Adiantum capillus-veneris. We detected 350 RNA editing sites when the cDNA sequence was compared to that of the genomic DNA. Of these sites, 10% were U-to-C edits and 90% were C-to-U edits. RNA editing created 19 new start codons, three new stop codons, and "repaired" 26 internal stop codons. Of the 332 editing sites that altered a codon, 26% were in the first codon position, 68% in the second, and 6% in the third. We also detected 21 silent edits, as well as 19 edits that were in untranslated regions, including introns and the anticodon of tRNA(Leu). The latter edit provided a tRNA that is not otherwise encoded in this genome and accounts for a heavily used leucine codon. The level of RNA editing in this fern is more than ten times that of any other vascular plant examined across an entire chloroplast genome. A previous study found even higher levels of editing in a hornwort (942 sites). This suggests that the relatively low levels of editing in seed plants (less than 0.05%) may not be typical for land plants, and that RNA editing may play a major role in chloroplast genome processing. Additionally, we found that 53 editing sites in A. capillus-veneris are homologous to editing sites in the hornwort, and some other land plants. This implies that a major component of RNA editing sites have been conserved for hundreds of millions of years.

Adiantum↗

Prediction of glycosylphosphatidylinositol-anchored proteins in Arabidopsis. A genomic analysis.

Glycosylphosphatidylinositol (GPI) anchoring of proteins provides a potential mechanism for targeting to the plant plasma membrane and cell wall. However, relatively few such proteins have been identified. Here, we develop a procedure for database analysis to identify GPI-anchored proteins (GAP) based on their possession of common features. In a comprehensive search of the annotated Arabidopsis genome, we identified 167 novel putative GAP in addition to the 43 previously described candidates. Many of these 210 proteins show similarity to characterized cell surface proteins. The predicted GAP include homologs of beta-1,3-glucanases (16), metallo- and aspartyl proteases (13), glycerophosphodiesterases (6), phytocyanins (25), multi-copper oxidases (2), extensins (6), plasma membrane receptors (19), and lipid-transfer-proteins (18). Classical arabinogalactan (AG) proteins (13), AG peptides (9), fasciclin-like proteins (20), COBRA and 10 homologs, and novel potential signaling peptides that we name GAPEPs (8) were also identified. A further 34 proteins of unknown function were predicted to be GPI anchored. A surprising finding was that over 40% of the proteins identified here have probable AG glycosylation modules, suggesting that AG glycosylation of cell surface proteins is widespread. This analysis shows that GPI anchoring is likely to be a major modification in plants that is used to target a specific subset of proteins to the cell surface for extracellular matrix remodeling and signaling.

Arabidopsis↗