BackgroundAlthough the genome sequence of the protozoan parasite Leishmania major was determined several years ago, the knowledge of its transcriptome was incomplete, both regarding the real number of genes and their primary structure.ResultsHere, we describe the first comprehensive transcriptome analysis of a parasite from the genus Leishmania. Using high-throughput RNA sequencing (RNA-seq), a total of 10285 transcripts were identified, of which 1884 were considered novel, as they did not match previously annotated genes. In addition, our data indicate that current annotations should be modified for many of the genes. The detailed analysis of the transcript processing sites revealed extensive heterogeneity in the spliced leader (SL) and polyadenylation addition sites. As a result, around 50% of the genes presented multiple transcripts differing in the length of the UTRs, sometimes in the order of hundreds of nucleotides. This transcript heterogeneity could provide an additional source for regulation as the different sizes of UTRs could modify RNA stability and/or influence the efficiency of RNA translation. In addition, for the first time for the Leishmania major promastigote stage, we are providing relative expression transcript levels.ConclusionsThis study provides a concise view of the global transcriptome of the L. major promastigote stage, providing the basis for future comparative analysis with other development stages or other Leishmania species.
SummaryThe successful application of the maize transposable element system AciDs as a genome mutagen in heterologous plant species has recently proved the versatility and power of this technique in plant molecular biology. However, the frequency of Ac/Ds transposition is considerably lower in Arabidopsis thaliana than in most other dicot plant species that have been studied. Since previous research has established that transcripts derived from monocot genes can be alternatively processed in dicot plants, we have investigated both the efficiency of intron splicing and poiyadenylation of the maize Ac transposase pre-mRNA in Arabidopsis thaliana, Nicotiana tabacum, Nicotiana plumbaginifolia and Zea mays. In this paper, we demonstrate that intron 4 is alternatively spliced within Arabidopsis, using cryptic 5' and 3' splice sites within the intron sequence, leading to a heterogeneous population of full length transposase transcript. Furthermore, analysis of transposase transcript polyadenylation revealed that at least four alternative poly(A) sites were utilized between introns 2 and 3, resulting in truncated transposase transcripts. Finally, by Northern blotting, we established that the truncated transposase transcript was the most abundant form of transposase message in Arabidopsis. In contrast to these findings, the alternative splicing and premature polyadenylation of Ac message in Arabidopsis was unparalleled in the other species examined. We suggest that the poor frequency of transposition of Ac in Arabidopsis may be in part due to the low quantity of correctly processed transposase transcript available in this species.
As many tumor cells synthetize vascular endothelial growth factors (VEGF) that promote neo-vascularization and metastasis, frontline cancer therapies often administer anti-VEGF (α-VEGF) antibodies. To target the oncolytic parvovirus minute virus of mice (MVM) to the tumor vasculature, we studied the functional tolerance, evasion of neutralization, and induction of α-VEGF antibodies of chimeric viruses in which the footprint of a neutralizing monoclonal antibody within the 3-fold capsid spike was replaced by VEGF-blocking peptides: P6L (PQPRPL) and A7R (ATWLPPR). Both peptides allowed viral genome replication and nuclear translocation of chimeric capsid subunits. MVM-P6L efficiently propagated in culture, exposing the heterologous peptide on the capsid surface, and evaded neutralization by the anti-spike monoclonal antibody. In contrast, MVM-A7R yielded low infectious titers and was poorly recognized by an α-A7R monoclonal antibody. MVM-A7R showed a deficient assembly pattern, suggesting that A7R impaired a transitional configuration that the subunits must undergo in the 3-fold axis to close up the capsid shell. The MVM-A7R chimeric virus consistently evolved in culture into a mutant carrying the P6Q amino acid substitution within the A7R sequence, which restored normal capsid assembly and infectivity. Consistent with this finding, anti-native VEGF antibodies were induced in mice by a single injection of MVM-A7R empty capsids, but not by MVM-A7R virions. This fundamental study provides insights to endow an infectious parvovirus with immune antineovascularization and evasion capacities by replacing an antibody footprint in the capsid 3-fold axis with VEGF-blocking peptides, and it also illustrates the evolutionary capacity of single-stranded DNA (ssDNA) viruses to overcome engineered capsid structural restrictions.IMPORTANCETargeting the VEGF signaling required for neovascularization by vaccination with chimeric capsids of oncolytic viruses may boost therapy for solid tumors. VEGF-blocking peptides (VEbp) engineered in the capsid 3-fold axis endowed the infectious parvovirus MVM with the ability to induce α-VEGF antibodies without adjuvant and to evade neutralization by MVM-specific antibodies. However, these properties may be compromised by structural restraints that the capsid imposes on the peptide configuration and by misassembly caused by the heterologous peptides. Significantly, chimeric MVM-VEbp resolved the structural restrictions by selecting mutations within the engineered peptides that restored efficient capsid assembly. These data show the promise of antineovascularization vaccines using chimeric VEbp-icosahedral capsids of oncolytic viruses but also raise safety concerns regarding the genetic stability of manipulated infectious parvoviruses in cancer and gene therapies.
Human herpesviruses 6A and 6B (HHV-6A/6B) are ubiquitous pathogens that persist lifelong in latent form and can cause severe conditions upon reactivation. They are spread by community-acquired infection of free virus (acqHHV6A/6B) and by germline transmission of inherited chromosomally-integrated HHV-6A/6B (iciHHV-6A/6B) in telomeres. We exploited a hypervariable region of the HHV-6B genome to investigate the relationship between acquired and inherited virus and revealed predominantly maternal transmission of acqHHV-6B in families. Remarkably, we demonstrate that some copies of acqHHV-6B in saliva from healthy adults gained a telomere, indicative of integration and latency, and that the frequency of viral genome excision from telomeres in iciHHV-6B carriers is surprisingly high and varies between tissues. In addition, newly formed short telomeres generated by partial viral genome release are frequently lengthened, particularly in telomerase-expressing pluripotent cells. Consequently, iciHHV-6B carriers are mosaic for different iciHHV-6B structures, including circular extra-chromosomal forms that have the potential to reactivate. Finally, we show transmission of an HHV-6B strain from an iciHHV-6B mother to her non-iciHHV-6B son. Altogether we demonstrate that iciHHV-6B can readily transition between telomere-integrated and free virus forms.
BackgroundCopy number variation (CNV) is a major component of genomic variation, yet methods to accurately type genomic CNV lag behind methods that type single nucleotide variation. High-throughput sequencing can contribute to these methods by using sequence read depth, which takes the number of reads that map to a given part of the reference genome as a proxy for copy number of that region, and compares across samples. Furthermore, high-throughput sequencing also provides information on the sequence differences between copies within and between individuals.MethodsIn this study we use high-coverage phase 3 exome sequences of the 1000 Genomes project to infer diploid copy number of the beta-defensin genomic region, a well-studied CNV that carries several beta-defensin genes involved in the antimicrobial response, signalling, and fertility. We also use these data to call sequence variants, a particular challenge given the multicopy nature of the region.ResultsWe confidently call copy number and sequence variation of the beta-defensin genes on 1285 samples from 26 global populations, validate copy number using Nanostring nCounter and triplex paralogue ratio test data. We use the copy number calls to verify the genomic extent of the CNV and validate sequence calls using analysis of cloned PCR products. We identify novel variation, mostly individually rare, predicted to alter amino-acid sequence in the beta-defensin genes. Such novel variants may alter antimicrobial properties or have off-target receptor interactions, and may contribute to individuality in immunological response and fertility.ConclusionsGiven that 81 % of identified sequence variants were not previously in dbSNP, we show that sequence variation in multiallelic CNVs represent an unappreciated source of genomic diversity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2123-y) contains supplementary material, which is available to authorized users.
Aim: To further investigate the postulated synergistic interaction between the insertioddeletion (UD) polymorphism within the angiotensin-converting enzyme (ACE) gene and an AIC transversion at nucleotide position 1166 within the angiotensin I1 sub-type I receptor (ATIR) gene on risk of myocardial infarction (MI). Methods: 541 patients were recruited at the time of M1 from the coronary care units (CCLJ) of the Leicester Royal lntirmary (7/93-
Human herpesviruses 6A and 6B (HHV-6A/6B) are ubiquitous pathogens that persist lifelong in latent form and can cause severe conditions upon reactivation. They are spread by community-acquired infection of free virus (acqHHV6A/6B) and by germline transmission of inherited chromosomally-integrated HHV-6A/6B (iciHHV-6A/6B) in telomeres. We exploited a hypervariable region of the HHV-6B genome to investigate the relationship between acquired and inherited virus and revealed predominantly maternal transmission of acqHHV-6B in families. Remarkably, we demonstrate that some copies of acqHHV-6B in saliva from healthy adults gained a telomere, indicative of integration and latency, and that the frequency of viral genome excision from telomeres in iciHHV-6B carriers is surprisingly high and varies between tissues. In addition, newly formed short telomeres generated by partial viral genome release are frequently lengthened, particularly in telomerase-expressing pluripotent cells. Consequently, iciHHV-6B carriers are mosaic for different iciHHV-6B structures, including circular extra-chromosomal forms that have the potential to reactivate. Finally, we show transmission of an HHV-6B strain from an iciHHV-6B mother to her non-iciHHV-6B son. Altogether we demonstrate that iciHHV-6B can readily transition between telomere-integrated and free virus forms.
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