Distinct Genome Organization Research Articles

Characterization of the mitochondrial Huso huso genome and new aspects of its organization in the presence of tandem repeats in 12S rRNA

BackgroundThe sturgeon group has been economically significant worldwide due to caviar production. Sturgeons consist of 27 species in the world. Mitogenome data could be used to infer genetic diversity and investigate the evolutionary history of sturgeons. A limited number of complete mitogenomes in this family were sequenced. Here, we annotated the mitochondrial Huso huso genome, which revealed new aspects of this species.ResultsIn this species, the mitochondrial genome consisted of 13 genes encoding proteins, 22tRNA and 2rRNA, and two non-coding regions that followed other vertebrates. In addition, H. huso had a pseudo-tRNA-Glu between ND6 and Cytb and a 52-nucleotide tandem repeat with two replications in 12S rRNA. This duplication event is probably related to the slipped strand during replication, which could remain in the strand due to mispairing during replication. Furthermore, an 82 bp repeat sequence with three replications was observed in the D-loop control region, which is usually visible in different species. Regulatory elements were also seen in the control region of the mitochondrial genome, which included termination sequences and conserved regulatory blocks. Genomic compounds showed the highest conservation in rRNA and tRNA, while protein-encoded genes and nonencoded regions had the highest divergence. The mitochondrial genome was phylogenetically assayed using 12 protein-encoding genes.ConclusionsIn H. huso sequencing, we identified a distinct genome organization relative to other species that have never been reported. In recent years, along with the advancement in sequencing identified more genome rearrangements. However, it is an essential aspect of researching the evolution of the mitochondrial genome that needs to be recognized.

Uncovering Pseudogenes and Intergenic Protein-coding Sequences in TriTryps' Genomes.

Trypanosomatids belong to a remarkable group of unicellular, parasitic organisms of the order Kinetoplastida, an early diverging branch of the phylogenetic tree of eukaryotes, exhibiting intriguing biological characteristics affecting gene expression (intronless polycistronic transcription, trans-splicing, and RNA editing), metabolism, surface molecules, and organelles (compartmentalization of glycolysis, variation of the surface molecules, and unique mitochondrial DNA), cell biology and life cycle (phagocytic vacuoles evasion and intricate patterns of cell morphogenesis). With numerous genomic-scale data of several trypanosomatids becoming available since 2005 (genomes, transcriptomes, and proteomes), the scientific community can further investigate the mechanisms underlying these unusual features and address other unexplored phenomena possibly revealing biological aspects of the early evolution of eukaryotes. One fundamental aspect comprises the processes and mechanisms involved in the acquisition and loss of genes throughout the evolutionary history of these primitive microorganisms. Here, we present a comprehensive in silico analysis of pseudogenes in three major representatives of this group: Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. Pseudogenes, DNA segments originating from altered genes that lost their original function, are genomic relics that can offer an essential record of the evolutionary history of functional genes, as well as clues about the dynamics and evolution of hosting genomes. Scanning these genomes with functional proteins as proxies to reveal intergenic regions with protein-coding features, relying on a customized threshold to distinguish statistically and biologically significant sequence similarities, and reassembling remnant sequences from their debris, we found thousands of pseudogenes and hundreds of open reading frames, with particular characteristics in each trypanosomatid: mutation profile, number, content, density, codon bias, average size, single- or multi-copy gene origin, number and type of mutations, putative primitive function, and transcriptional activity. These features suggest a common process of pseudogene formation, different patterns of pseudogene evolution and extant biological functions, and/or distinct genome organization undertaken by those parasites during evolution, as well as different evolutionary and/or selective pressures acting on distinct lineages.

A short review on sugarcane: its domestication, molecular manipulations and future perspectives.

Sugarcane (Saccharum spp.) is a special crop plant that underwent anthropogenic evolution from a wild grass species to an important food, fodder, and energy crop. Unlike any other grass species which were selected for their kernels, sugarcane was selected for its high stem sucrose accumulation. Flowering in sugarcane is not favored since flowering diverts the stored sugar resources for the reproductive and developmental energy needs. Cultivars are vegetatively propagated and sugarcane breeding is still essentially focused on conventional methods, since the knowledge of sugarcane genetics has lagged that of other major crops. Cultivar improvement has been extremely challenging due to its polyploidy and aneuploidy nature derived from a few interspecific hybridizations between Saccharum officinarum and Saccharum spontaneum, revealing the coexistence of two distinct genome organization modes in the modern variety. Alongside implementation of modern agricultural techniques, generation of hybrid clones, transgenics and genome edited events will help to meet the ever-growing bioenergy needs. Additionally, there are two common biotechnological approaches to improve plant stress tolerance, which includes marker-assisted selection (MAS) and genetic transformation. During the past two decades, the use of molecular approaches has contributed greatly to a better understanding of the genetic and biochemical basis of plant stress-tolerance and in some cases, it led to the development of plants with enhanced tolerance to abiotic stress. Hence, this review mainly intends on the events that shaped the sugarcane as what it is now and what challenges ahead and measures taken to further improve its yield, production and maximize utilization to beat the growing demands.

Sexual reproduction contributes to the evolution of resistance-breaking isolates of the spinach pathogen Peronospora effusa.

Summary Peronospora effusa causes downy mildew, the economically most important disease of cultivated spinach worldwide. To date, 19 P. effusa races have been denominated based on their capacity to break spinach resistances, but their genetic diversity and the evolutionary processes that contribute to race emergence are unknown. Here, we performed the first systematic analysis of P. effusa races showing that those emerge by both asexual and sexual reproduction. Specifically, we studied the diversity of 26 P. effusa isolates from 16 denominated races based on mitochondrial and nuclear comparative genomics. Mitochondrial genomes based on long‐read sequencing coupled with diversity assessment based on short‐read sequencing uncovered two mitochondrial haplogroups, each with distinct genome organization. Nuclear genome‐wide comparisons of the 26 isolates revealed that 10 isolates from six races could clearly be divided into three asexually evolving groups, in concordance with their mitochondrial phylogeny. The remaining isolates showed signals of reticulated evolution and discordance between nuclear and mitochondrial phylogenies, suggesting that these evolved through sexual reproduction. Increased understanding of this pathogen's reproductive modes will provide the framework for future studies into the molecular mechanisms underlying race emergence and into the P. effusa‐spinach interaction, thus assisting in sustainable production of spinach through knowledge‐driven resistance breeding.

Biological and Molecular Characterization of Two Closely Related Arepaviruses and Their Antagonistic Interaction in Nicotiana benthamiana.

Previously, our group characterized two closely related viruses from Areca catechu, areca palm necrotic ringspot virus (ANRSV) and areca palm necrotic spindle-spot virus (ANSSV). These two viruses share a distinct genomic organization of leader proteases and represent the only two species of the newly established genus Arepavirus of the family Potyviridae. The biological features of the two viruses are largely unknown. In this study, we investigated the pathological properties, functional compatibility of viral elements, and interspecies interactions in the model plant, Nicotiana benthamiana. Using a newly obtained infectious clone of ANRSV, we showed that this virus induces more severe symptoms compared with ANSSV and that this is related to a rapid virus multiplication in planta. A series of hybrid viruses were constructed via the substitution of multiple elements in the ANRSV infectious clone with the counterparts of ANSSV. The replacement of either 5′-UTR-HCPro1–HCPro2 or CI effectively supported replication and systemic infection of ANRSV, whereas individual substitution of P3-7K, 9K-NIa, and NIb-CP-3′-UTR abolished viral infectivity. Finally, we demonstrated that ANRSV confers effective exclusion of ANSSV both in coinfection and super-infection assays. These results advance our understanding of fundamental aspects of these two distinct but closely related arepaviruses.

Genome Sequences of Serratia Strains Revealed Common Genes in Both Serratomolides Gene Clusters.

Simple SummaryBiosurfactants are amphiphilic molecules produced by microorganisms with a hydrophilic and a hydrophobic group, able to reduce surface tension. These molecules are largely used in the environmental, food, pharmaceutical, medical, and cleaning industries, among others. Serratia strains are ubiquitous microorganisms with the ability to produce biosurfactants, such as serrawettins. These extracellular lipopeptides are described as biocides against many bacteria and fungi. This work used comparative genomics to determine the distribution and organization of the serrawettins W1 and W2 biosynthetic gene clusters in all the 84 publicly available genomes of the Serratia genus. Here, the serrawettin W1 gene clusters’ organization is reported for the first time. The serrawettin W1 biosynthetic gene swrW and serrawettin W2 biosynthetic gene swrA were present in 17 and 11 Serratia genomes, respectively. The same genes in the biosynthetic clusters frame the swrW and swrA biosynthetic genes. This work identified four genes common to all serrawettin gene clusters, highlighting their key potential in the serrawettins biosynthetic process.Serratia strains are ubiquitous microorganisms with the ability to produce serratomolides, such as serrawettins. These extracellular lipopeptides are described as biocides against many bacteria and fungi and may have a nematicidal activity against phytopathogenic nematodes. Serrawettins W1 and W2 from different strains have different structures that might be correlated with distinct genomic organizations. This work used comparative genomics to determine the distribution and the organization of the serrawettins biosynthetic gene clusters in all the 84 publicly available genomes of the Serratia genus. The serrawettin W1 and W2 gene clusters’ organization was established using antiSMASH software and compared with single and short data previously described for YD25T Serratia. Here, the serrawettin W1 gene clusters’ organization is reported for the first time. The serrawettin W1 biosynthetic gene swrW was present in 17 Serratia genomes. Eighty different coding sequence (CDS) were assigned to the W1 gene cluster, 13 being common to all clusters. The serrawettin W2 swrA gene was present in 11 Serratia genomes. The W2 gene clusters included 68 CDS with 24 present in all the clusters. The genomic analysis showed the swrA gene constitutes five modules, four with three domains and one with four domains, while the swrW gene constitutes one module with four domains. This work identified four genes common to all serrawettin gene clusters, highlighting their essential potential in the serrawettins biosynthetic process.

Molecular Characterization of Hovenia Dulcis-Associated Virus 1 (HDaV1) and 2 (HDaV2): New Tentative Species within the Order Picornavirales

In a systematic field survey for plant-infecting viruses, leaf tissues were collected from trees showing virus-like symptoms in Brazil. After viral enrichment, total RNA was extracted and sequenced using the MiSeq platform (Illumina). Two nearly full-length picorna-like genomes of 9534 and 8158 nucleotides were found associated with Hovenia dulcis (Rhamnaceae family). Based upon their genomic information, specific primers were synthetized and used in RT-PCR assays to identify plants hosting the viral sequences. The larger contig was tentatively named as Hovenia dulcis-associated virus 1 (HDaV1), and it exhibited low nucleotide and amino acid identities with Picornavirales species. The smaller contig was related to insect-associated members of the Dicistroviridae family but exhibited a distinct genome organization with three non-overlapping open reading frames (ORFs), and it was tentatively named as Hovenia dulcis-associated virus 2 (HDaV2). Phylogenetic analysis using the amino acid sequence of RNA-dependent RNA polymerase (RdRp) revealed that HDaV1 and HDaV2 clustered in distinct groups, and both viruses were tentatively assigned as new members of the order Picornavirales. HDaV2 was assigned as a novel species in the Dicistroviridae family. The 5′ ends of both viruses are incomplete. In addition, a nucleotide composition analysis (NCA) revealed that HDaV1 and HDaV2 have similarities with invertebrate-infecting viruses, suggesting that the primary host(s) of these novel virus species remains to be discovered.

Geminiviruses: Taxonomic Structure and Diversity in Genomic Organization.

Geminiviridae is one of the best-characterized and hence, one of the largest plant-virus families with the highest economic importance. Its members characteristically have a circular ssDNA genome within the encapsidation of twinned quasi-icosaheadral virions (18-38 nm size-range). Construction of a narrative review on geminiviruses, to have a clearer picture of their genomic structure and taxonomic status. A thorough search was conducted for papers and patents regarding geminiviruses, where relevant information was used to study their genomic organization, diversity and taxonomic structure. Geminiviruses have been classified into nine genera (viz., genus Begomovirus, Mastrevirus, Curtovirus, Topocuvirus, Becurtovirus, Turncurtovirus, Capulavirus, Eragrovirus and Grablovirus) having distinct genomic organizations, host ranges and insect vectors. Genomic organization of all genera generally shows the presence of 4-6 ORFs encoding for various proteins. For now, Citrus chlorotic dwarf-associated virus (CCDaV), Camellia chlorotic dwarf-associated virus (CaCDaV) and few other geminiviruses are still unassigned to any genera. The monopartite begomoviruses (and few mastreviruses) have been found associated with aplhasatellites and betasatellites (viz., ~1.3 kb circular ssDNA satellites). Recent reports suggest that deltasatellites potentially reduce the accumulation of helper-Begomovirus species in host plants. Some patents have revealed the methods to generate transgenic plants resistant to geminiviruses. Geminiviruses rapidly evolve and are a highly diverse group of plant-viruses. However, research has shown new horizons in tackling the acute begomoviral diseases in plants by generating a novel bio-control methodology in which deltasatellites can be used as bio-control agents and generate transgenic plants resistant to geminiviruses.

Complete nucleotide sequence of loquat virus A, a member of the family Betaflexiviridae with a novel genome organization.

Analysis of a loquat tree with leaf curl symptoms by deep sequencing revealed a novel virus with a single-stranded RNA genome, for which the name "loquat virus A" (LoVA) was proposed. The complete genome sequence comprised 7553 nucleotides (nt) and an additional poly(A) tail at the 3' terminus. Sequence comparisons of LoVA showed moderate similarity to cherry virus A (CVA), currant virus A (CuVA), and mume virus A (MuVA), which are members of the genus Capillovirus in the family Betaflexiviridae. Phylogenetic analysis of the full-genome nt sequence and replicase-like protein supported the placement of LoVA within the genus Capillovirus. However, it has a distinct genome organization, differing from recognized capilloviruses, as it contains three open reading frames (ORFs), with the coat protein (CP) expressed separately from the replication-associated protein (RP) rather than being encoded in the same ORF. This indicates that LoVA is a novel member of the genus Capillovirus in the family Betaflexiviridae with a distinct genomic organization.

An isolate of sweet potato chlorotic stunt virus from Brazil with a distinct genome organization.

Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae), is an economically important pathogen of sweet potato. In the present work, the nucleotide sequences of two RNA segments of SPCSV (isolate SPCSV-UNB-01) were determined by MiSeq Illumina sequencing of samples of sweet potato plants grafted onto Ipomoea setosa. A comparative analysis of the genome organization of SPCSV-UNB-01 and other SPCSV sequences showed that RNA1 was lacking p22, and p5.1 and that p5.2. was absent in RNA2, indicating a unique genomic pattern. SPCSV-UNB-01 contained longer p6 and p5 regions, with little similarity to orthologous sequences. Sequence comparison did not reveal any previously identified functional domains within these open reading frames (ORFs). No recombination or rearrangement events were detected. Phylogenetic analysis suggested the possibility of separate entries of SPCSV into South America based on the genetic distance between SPCSV-UNB-01 and the Peruvian isolate m2-47. Samples from northeastern Brazil (State of Pernambuco) were positive for SPCSV when tested using specific primers for the major coat protein (CP) gene. This is the first full-length genome sequence of SPCSV-UNB-01 from Brazil.

Mitochondrial genome organization varies among different groups of the booklouse, Liposcelis bostrychophila :

The booklouse, Liposcelis bostrychophila is an important stored pest worldwide. The mt genome of an asexual strain (Beibei, China) of the booklouse, L. bostrychophila, comprises two chromosomes; each chromosome contains approximate half of the 37 genes typically found in animals. The mt genomes of two sexual strains of L. bostrychophila, however, comprise five and seven chromosomes respectively; each chromosome contains one to six genes. To understand mt genome evolution in L. bostrychophila, we sequenced the mt genomes of six strains of asexual L. bostrychophila collected from different locations in China, Croatia and USA. The mt genomes of all of the six asexual strains of L. bostrychophila collected in China, Croatia and USA have two chromosomes. Phylogenetic analysis of mt genome sequences divided nine strains of L. bostrychophila into four groups. Each group has a distinct mt genome organization and substantial sequence divergence (48.7-87.4%) from other groups. Furthermore, the seven asexual strains of L. bostrychophila including the published Beibei strain are more closely related to two other species of booklice, L. paeta and L. sculptilis, than to the sexual strains of L. bostrychophila. Our results revealed highly divergent mt genomes in the booklouse, L. bostrychophila, and indicated that L. bostrychophila is a cryptic species.

A Virus in American Blackcurrant (Ribes americanum) with Distinct Genome Features Reshapes Classification in the Tymovirales.

A novel virus with distinct genome features was discovered by high throughput sequencing in a symptomatic blackcurrant plant. The virus, tentatively named Ribes americanum virus A (RAVA), has distinct genome organization and molecular features bridging genera in the order Tymovirales. The genome consists of 7106 nucleotides excluding the poly(A) tail. Five open reading frames were identified, with the first encoding a putative viral replicase with methyl transferase (MTR), AlkB, helicase, and RNA dependent RNA polymerase (RdRp) domains. The genome organization downstream of the replicase resembles that of members of the order Tymovirales with an unconventional triple gene block (TGB) movement protein arrangement with none of the other four putative proteins exhibiting significant homology to viral proteins. Phylogenetic analysis using replicase conserved motifs loosely placed RAVA within the Betaflexiviridae. Data strongly suggest that RAVA is a novel virus that should be classified as a species in a new genus in the Betaflexiviridae or a new family within the order Tymovirales.

The Highly Divergent Mitochondrial Genomes Indicate That the Booklouse, Liposcelis bostrychophila (Psocoptera: Liposcelididae) Is a Cryptic Species.

The booklouse, Liposcelis bostrychophila is an important storage pest worldwide. The mitochondrial (mt) genome of an asexual strain (Beibei, China) of the L. bostrychophila comprises two chromosomes; each chromosome contains approximate half of the 37 genes typically found in bilateral animals. The mt genomes of two sexual strains of L. bostrychophila, however, comprise five and seven chromosomes, respectively; each chromosome contains one to six genes. To understand mt genome evolution in L. bostrychophila, and whether L. bostrychophila is a cryptic species, we sequenced the mt genomes of six strains of asexual L. bostrychophila collected from different locations in China, Croatia, and the United States. The mt genomes of all six asexual strains of L. bostrychophila have two chromosomes. Phylogenetic analysis of mt genome sequences divided nine strains of L. bostrychophila into four groups. Each group has a distinct mt genome organization and substantial sequence divergence (48.7–87.4%) from other groups. Furthermore, the seven asexual strains of L. bostrychophila, including the published Beibei strain, are more closely related to two other species of booklice, L. paeta and L. sculptilimacula, than to the sexual strains of L. bostrychophila. Our results revealed highly divergent mt genomes in the booklouse, L. bostrychophila, and indicate that L. bostrychophila is a cryptic species.

Campylobacter group II phage CP21 is the prototype of a new subgroup revealing a distinct modular genome organization and host specificity

BackgroundThe application of phages is a promising tool to reduce the number of Campylobacter along the food chain. Besides the efficacy against a broad range of strains, phages have to be safe in terms of their genomes. Thus far, no genes with pathogenic potential (e.g., genes encoding virulence factors) have been detected in Campylobacter phages. However, preliminary studies suggested that the genomes of group II phages may be diverse and prone to genomic rearrangements.ResultsWe determined and analysed the genomic sequence (182,761 bp) of group II phage CP21 that is closely related to the already characterized group II phages CP220 and CPt10. The genomes of these phages are comprised of four modules separated by very similar repeat regions, some of which harbouring open reading frames (ORFs). Though, the arrangement of the modules and the location of some ORFs on the genomes are different in CP21 and in CP220/CPt10. In this work, a PCR system was established to study the modular genome organization of other group II phages demonstrating that they belong to different subgroups of the CP220-like virus genus, the prototypes of which are CP21 and CP220. The subgroups revealed different restriction patterns and, interestingly enough, also distinct host specificities, tail fiber proteins and tRNA genes. We additionally analysed the genome of group II phage vB_CcoM-IBB_35 (IBB_35) for which to date only five individual contigs could be determined. We show that the contigs represent modules linked by long repeat regions enclosing some yet not identified ORFs (e.g., for a head completion protein). The data suggest that IBB_35 is a member of the CP220 subgroup.ConclusionCampylobacter group II phages are diverse regarding their genome organization. Since all hitherto characterized group II phages contain numerous genes for transposases and homing endonucleases as well as similar repeat regions, it cannot be excluded that these phages are genetically unstable. To answer this question, further experiments and sequencing of more group II phages should be performed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1837-1) contains supplementary material, which is available to authorized users.

U exon variability as a discriminatory tool for Bayesian analysis of adenoviruses

Members of the family Adenoviridae are divided into five genera and infect a wide variety of vertebrates with a narrow host range, usually restricted to one species. Due to the high genetic diversity and distinct genomic organization, classification of adenoviruses is difficult to achieve and often performed by phylogenetic analysis. The most commonly used region for phylogenetic inference of adenoviruses is the DNA polymerase (AdPol) gene carried out at amino acid level. In this paper we investigated the suitability of the U exon to discriminate adenoviruses. The tree based on this genus-common feature, obtained with 23 short amino acid sequences, offered a clearest discrimination of the members of the adenovirus family (Adenoviridae) than the trees generated with the complete or partial polymerase protein sequences. Therefore, our results demonstrate that the U exon is an effective tool for a refined phylogenetic inference and genus classification of the Adenoviridae family.

Reconstruction of the Ancestral Plastid Genome in Geraniaceae Reveals a Correlation between Genome Rearrangements, Repeats, and Nucleotide Substitution Rates

Geraniaceae plastid genomes are highly rearranged, and each of the four genera already sequenced in the family has a distinct genome organization. This study reports plastid genome sequences of six additional species, Francoa sonchifolia, Melianthus villosus, and Viviania marifolia from Geraniales, and Pelargonium alternans, California macrophylla, and Hypseocharis bilobata from Geraniaceae. These genome sequences, combined with previously published species, provide sufficient taxon sampling to reconstruct the ancestral plastid genome organization of Geraniaceae and the rearrangements unique to each genus. The ancestral plastid genome of Geraniaceae has a 4 kb inversion and a reduced, Pelargonium-like small single copy region. Our ancestral genome reconstruction suggests that a few minor rearrangements occurred in the stem branch of Geraniaceae followed by independent rearrangements in each genus. The genomic comparison demonstrates that a series of inverted repeat boundary shifts and inversions played a major role in shaping genome organization in the family. The distribution of repeats is strongly associated with breakpoints in the rearranged genomes, and the proportion and the number of large repeats (>20 bp and >60 bp) are significantly correlated with the degree of genome rearrangements. Increases in the degree of plastid genome rearrangements are correlated with the acceleration in nonsynonymous substitution rates (dN) but not with synonymous substitution rates (dS). Possible mechanisms that might contribute to this correlation, including DNA repair system and selection, are discussed.

Plastome Sequences of Lygodium japonicum and Marsilea crenata Reveal the Genome Organization Transformation from Basal Ferns to Core Leptosporangiates

Previous studies have shown that core leptosporangiates, the most species-rich group of extant ferns (monilophytes), have a distinct plastid genome (plastome) organization pattern from basal fern lineages. However, the details of genome structure transformation from ancestral ferns to core leptosporangiates remain unclear because of limited plastome data available. Here, we have determined the complete chloroplast genome sequences of Lygodium japonicum (Lygodiaceae), a member of schizaeoid ferns (Schizaeales), and Marsilea crenata (Marsileaceae), a representative of heterosporous ferns (Salviniales). The two species represent the sister and the basal lineages of core leptosporangiates, respectively, for which the plastome sequences are currently unavailable. Comparative genomic analysis of all sequenced fern plastomes reveals that the gene order of L. japonicum plastome occupies an intermediate position between that of basal ferns and core leptosporangiates. The two exons of the fern ndhB gene have a unique pattern of intragenic copy number variances. Specifically, the substitution rate heterogeneity between the two exons is congruent with their copy number changes, confirming the constraint role that inverted repeats may play on the substitution rate of chloroplast gene sequences.

Distinct genomic organization, mRNA expression and cellular localization of members of two amastin sub-families present in Trypanosoma cruzi

BackgroundAmastins are surface glycoproteins (approximately 180 residues long) initially described in Trypanosoma cruzi as particularly abundant during the amastigote stage of this protozoan parasite. Subsequently, they have been found to be encoded by large gene families also present in the genomes of several species of Leishmania and in other Trypanosomatids. Although most amastin genes are organized in clusters associated with tuzin genes and are up-regulated in the intracellular stage of T. cruzi and Leishmania spp, distinct genomic organizations and mRNA expression patterns have also been reported.ResultsBased on the analysis of the complete genome sequences of two T. cruzi strains, we identified a total of 14 copies of amastin genes in T. cruzi and showed that they belong to two of the four previously described amastin subfamilies. Whereas δ-amastin genes are organized in two or more clusters with alternating copies of tuzin genes, the two copies of β-amastins are linked together in a distinct chromosome. Most T. cruzi amastins have similar surface localization as determined by confocal microscopy and western blot analyses. Transcript levels for δ-amastins were found to be up-regulated in amastigotes from several T. cruzi strains, except in the G strain, which is known to have low infection capacity. In contrast, in all strains analysed, β-amastin transcripts are more abundant in epimastigotes, the stage found in the insect vector.ConclusionsHere we showed that not only the number and diversity of T. cruzi amastin genes is larger than what has been predicted, but also their mode of expression during the parasite life cycle is more complex. Although most T. cruzi amastins have a similar surface localization, only δ-amastin genes have their expression up-regulated in amastigotes. The results showing that a sub-group of this family is up-regulated in epimastigotes, suggest that, in addition of their role in intracellular amastigotes, T. cruzi amastins may also serve important functions during the insect stage of the parasite life cycle. Most importantly, evidence for their role as virulence factors was also unveiled from the data showing that δ-amastin expression is down regulated in a strain presenting low infection capacity.

Novel Insights into DNA Methylation Features in Spermatozoa: Stability and Peculiarities

Data about the entire sperm DNA methylome are limited to two sperm donors whereas studies dealing with a greater number of subjects focused only on a few genes or were based on low resolution arrays. This implies that information about what we can consider as a normal sperm DNA methylome and whether it is stable among different normozoospermic individuals is still missing. The definition of the DNA methylation profile of normozoospermic men, the entity of inter-individual variability and the epigenetic characterization of quality-fractioned sperm subpopulations in the same subject (intra-individual variability) are relevant for a better understanding of pathological conditions. We addressed these questions by using the high resolution Infinium 450K methylation array and compared normal sperm DNA methylomes against somatic and cancer cells. Our study, based on the largest number of subjects (n = 8) ever considered for such a large number of CpGs (n = 487,517), provided clear evidence for i) a highly conserved DNA methylation profile among normozoospermic subjects; ii) a stable sperm DNA methylation pattern in different quality-fractioned sperm populations of the same individual. The latter finding is particularly relevant if we consider that different quality fractioned sperm subpopulations show differences in their structural features, metabolic and genomic profiles. We demonstrate, for the first time, that DNA methylation in normozoospermic men remains highly uniform regardless the quality of sperm subpopulations. In addition, our analysis provided both confirmatory and novel data concerning the sperm DNA methylome, including its peculiar features in respect to somatic and cancer cells. Our description about a highly polarized sperm DNA methylation profile, the clearly distinct genomic and functional organization of hypo- versus hypermethylated loci as well as the association of histone-enriched hypomethylated loci with embryonic development, which we now extended also to hypomethylated piRNAs-linked genes, provides solid basis for future basic and clinical research.

Genomic sequencing and analyses of HearMNPV—a new Multinucleocapsid nucleopolyhedrovirus isolated from Helicoverpa armigera

BackgroundHearMNPV, a nucleopolyhedrovirus (NPV), which infects the cotton bollworm, Helicoverpa armigera, comprises multiple rod-shaped nucleocapsids in virion(as detected by electron microscopy). HearMNPV shows a different host range compared with H. armigera single-nucleocapsid NPV (HearSNPV). To better understand HearMNPV, the HearMNPV genome was sequenced and analyzed.MethodsThe morphology of HearMNPV was observed by electron microscope. The qPCR was used to determine the replication kinetics of HearMNPV infectious for H. armigera in vivo. A random genomic library of HearMNPV was constructed according to the “partial filling-in” method, the sequence and organization of the HearMNPV genome was analyzed and compared with sequence data from other baculoviruses.ResultsReal time qPCR showed that HearMNPV DNA replication included a decreasing phase, latent phase, exponential phase, and a stationary phase during infection of H. armigera. The HearMNPV genome consists of 154,196 base pairs, with a G + C content of 40.07%. 162 putative ORFs were detected in the HearMNPV genome, which represented 90.16% of the genome. The remaining 9.84% constitute four homologous regions and other non-coding regions. The gene content and gene arrangement in HearMNPV were most similar to those of Mamestra configurata NPV-B (MacoNPV-B), but was different to HearSNPV. Comparison of the genome of HearMNPV and MacoNPV-B suggested that HearMNPV has a deletion of a 5.4-kb fragment containing five ORFs. In addition, HearMNPV orf66, bro genes, and hrs are different to the corresponding parts of the MacoNPV-B genome.ConclusionsHearMNPV can replicate in vivo in H. armigera and in vitro, and is a new NPV isolate distinguished from HearSNPV. HearMNPV is most closely related to MacoNPV-B, but has a distinct genomic structure, content, and organization.