Intron Insertion Site Research Articles

Molecular characterization of a catalase gene from the green peach aphid (Myzus persicae).

The green peach aphid, Myzus persicae (Sulzer), is a serious agricultural pest with a worldwide distribution. Catalase (CAT), which is encoded by the catalase (Cat) gene, is an extremely important antioxidant enzyme that plays a pivotal role in protecting cells against the toxic effects of hydrogen peroxide. The Cat gene has not been characterized in M. persicae; therefore, this study describes the identification of the Cat (MpCat) gene from M. persicae. MpCat contains an open reading frame of 1515 bp and encodes a MpCAT protein consisting of 504 amino-acid residues. MpCAT possesses features typical of other insect catalases, including 7 conserved amino acids involved in binding heme and 15 involved in binding nicotinamide adenine dinucleotide phosphate. Phylogenetic analysis showed that MpCAT was closely related to orthologs from other aphid species. MpCat consisted of nine exons and eight introns, and the number and insertion sites of introns are consistent with those of Cat genes from Acyrthosiphon pisum (Harris) and Aphis gossypii Glover. The mRNA transcripts of MpCat were detected at all tested developmental stages, with the highest mRNA level in alate adults. The expression of MpCat was significantly upregulated when M. persicae was exposed to low and high temperatures, ultraviolet radiation, Beauveria bassiana, and permethrin. The transcription of MpCat and the activity of catalase were suppressed by RNA interference, and knockdown of MpCat significantly reduced the survival rate in M. persicae under heat stress. The results provide valuable information for further study on the physiological functions of MpCat.

Chloroplast Genomes of the Green-Tide Forming Alga Ulva compressa: Comparative Chloroplast Genomics in the Genus Ulva (Ulvophyceae, Chlorophyta)

To understand the evolution of Ulva chloroplast genomes at intraspecific and interspecific levels, in this study, three complete chloroplast genomes of Ulva compressa Linnaeus were sequenced and compared with the available Ulva cpDNA data. Our comparative analyses unveiled many noticeable findings. First, genome size variations of Ulva cpDNAs at intraspecific and interspecific levels were mainly caused by differences in gain or loss of group I/II introns, integration of foreign DNA fragments, and content of non-coding intergenic spacer regions. Second, chloroplast genomes of U. compressa shared the same 100 conserved genes as other Ulva cpDNA, whereas Ulva flexuosa appears to be the only Ulva species with the minD gene retained in its cpDNA. Third, five types of group I introns, most of which carry a LAGLIDADG or GIY-YIG homing endonuclease, and three of group II introns, usually encoding a reverse transcriptase/maturase, were detected at 26 insertion sites of 14 host genes in the 23 Ulva chloroplast genomes, and many intron insertion-sites have been found for the first time in Chlorophyta. Fourth, one degenerate group II intron previously ignored has been detected in the infA genes of all Ulva species, but not in the closest neighbor, Pseudoneochloris marina, and the other chlorophycean taxa, indicating that it should be the result of an independent invasion event that occurred in a common ancestor of Ulva species. Finally, the seven U. compressa cpDNAs represented a novel gene order which was different from that of other Ulva cpDNAs. The structure of Ulva chloroplast genomes is not conserved, but remarkably plastic, due to multiple rearrangement events.

Design of potentially universal SSU primers in myxomycetes using next-generation sequencing

Unlike fungi, which have a universally accepted barcode marker, universal primers still lack in myxomycetes. Typically, DNA barcode primers were designed based on comparing existing myxomycetes sequences and targeting the conserved regions. However, the extreme genetic diversity within major myxomycetes groups and the frequent occurrence of group I introns have made the development of universal DNA barcode a severe challenge. The emergence of next-generation sequencing provides an opportunity to address this problem. We sequenced the mixed genomic DNA of 81 myxomycetes and extracted the SSU gene's reads using next-generation sequencing. After alignment and assembly, we designed a set of SSU primers that matched all potential SNPs, avoided all known group I intron insertion sites, and were highly conserved between major myxomycetes orders. This set of SSU primers has the potential to become one of the universal primer combinations. Due to the high genetic divergence caused by long and complicated evolutionary histories, the lack of universal barcode primers is common in protists. Our research provides a new method to solve this problem.

Genome-Wide Survey and Expression Analysis of the KT/HAK/KUP Family in Brassica napus and Its Potential Roles in the Response to K+ Deficiency.

The KT/HAK/KUP (HAK) family is the largest potassium (K+) transporter family in plants, which plays key roles in K+ uptake and homeostasis, stress resistance, and root and embryo development. However, the HAK family has not yet been characterized in Brassica napus. In this study, 40 putative B. napus HAK genes (BnaHAKs) are identified and divided into four groups (Groups I–III and V) on the basis of phylogenetic analysis. Gene structure analysis revealed 10 conserved intron insertion sites across different groups. Collinearity analysis demonstrated that both allopolyploidization and small-scale duplication events contributed to the large expansion of BnaHAKs. Transcription factor (TF)-binding network construction, cis-element analysis, and microRNA prediction revealed that the expression of BnaHAKs is regulated by multiple factors. Analysis of RNA-sequencing data further revealed extensive expression profiles of the BnaHAKs in groups II, III, and V, with limited expression in group I. Compared with group I, most of the BnaHAKs in groups II, III, and V were more upregulated by hormone induction based on RNA-sequencing data. Reverse transcription-quantitative polymerase reaction analysis revealed that the expression of eight BnaHAKs of groups I and V was markedly upregulated under K+-deficiency treatment. Collectively, our results provide valuable information and key candidate genes for further functional studies of BnaHAKs.

Group II Introns Generate Functional Chimeric Relaxase Enzymes with Modified Specificities through Exon Shuffling at Both the RNA and DNA Level.

Group II introns are large self-splicing RNA enzymes with a broad but somewhat irregular phylogenetic distribution. These ancient retromobile elements are the proposed ancestors of approximately half the human genome, including the abundant spliceosomal introns and non-long terminal repeat retrotransposons. In contrast to their eukaryotic derivatives, bacterial group II introns have largely been considered as harmful selfish mobile retroelements that parasitize the genome of their host. As a challenge to this view, we recently uncovered a new intergenic trans-splicing pathway that generates an assortment of mRNA chimeras. The ability of group II introns to combine disparate mRNA fragments was proposed to increase the genetic diversity of the bacterial host by shuffling coding sequences. Here, we show that the Ll.LtrB and Ef.PcfG group II introns from Lactococcus lactis and Enterococcus faecalis respectively can both use the intergenic trans-splicing pathway to catalyze the formation of chimeric relaxase mRNAs and functional proteins. We demonstrated that some of these compound relaxase enzymes yield gain-of-function phenotypes, being significantly more efficient than their precursor wild-type enzymes at supporting bacterial conjugation. We also found that relaxase enzymes with shuffled functional domains are produced in biologically relevant settings under natural expression levels. Finally, we uncovered examples of lactococcal chimeric relaxase genes with junctions exactly at the intron insertion site. Overall, our work demonstrates that the genetic diversity generated by group II introns, at the RNA level by intergenic trans-splicing and at the DNA level by recombination, can yield new functional enzymes with shuffled exons, which can lead to gain-of-function phenotypes.

Comparative mitogenome analysis of two ectomycorrhizal fungi (Paxillus) reveals gene rearrangement, intron dynamics, and phylogeny of basidiomycetes

In this study, the mitogenomes of two Paxillus species were assembled, annotated and compared. The two mitogenomes of Paxillus involutus and P. rubicundulus comprised circular DNA molecules, with the size of 39,109 bp and 41,061 bp, respectively. Evolutionary analysis revealed that the nad4L gene had undergone strong positive selection in the two Paxillus species. In addition, 10.64 and 36.50% of the repetitive sequences were detected in the mitogenomes of P. involutus and P. rubicundulus, respectively, which might transfer between mitochondrial and nuclear genomes. Large-scale gene rearrangements and frequent intron gain/loss events were detected in 61 basidiomycete species, which revealed large variations in mitochondrial organization and size in Basidiomycota. In addition, the insertion sites of the basidiomycete introns were found to have a base preference. Phylogenetic analysis of the combined mitochondrial gene set gave identical and well-supported tree topologies, indicating that mitochondrial genes were reliable molecular markers for analyzing the phylogenetic relationships of Basidiomycota. This study is the first report on the mitogenomes of Paxillus, which will promote a better understanding of their contrasted ecological strategies, molecular evolution and phylogeny of these important ectomycorrhizal fungi and related basidiomycete species.

The fungal mitochondrial Nad5 pan-genic intron landscape

An intron landscape was prepared for the fungal mitochondrial nad5 gene. A hundred and eighty-eight fungal species were examined and a total of 265 introns were noted to be located in 29 intron insertion sites within the examined nad5 genes. Two hundred and sixty-three introns could be classified as group I types and two group II introns were noted. One additional group II intron module was identified nested within a composite group I intron. Based on features related to RNA secondary structures, introns can be classified into different subtypes and it was observed that intron insertion-sites are biased towards phase 0 and they appear to be specific to an intron type. Intron landscapes could be used as a guide map to predict the location of fungal mtDNA mobile introns, which are composite elements that include a ribozyme component and in some instances open reading frames encoding homing endonucleases or reverse transcriptases and all of these have applications in biotechnology.

Proposal of a new nomenclature for introns in protein-coding genes in fungal mitogenomes

Fungal mitochondrial genes are often invaded by group I or II introns, which represent an ideal marker for understanding fungal evolution. A standard nomenclature of mitochondrial introns is needed to avoid confusion when comparing different fungal mitogenomes. Currently, there has been a standard nomenclature for introns present in rRNA genes, but there is a lack of a standard nomenclature for introns present in protein-coding genes. In this study, we propose a new nomenclature system for introns in fungal mitochondrial protein-coding genes based on (1) three-letter abbreviation of host scientific name, (2) host gene name, (3), one capital letter P (for group I introns), S (for group II introns), or U (for introns with unknown types), and (4) intron insertion site in the host gene according to the cyclosporin-producing fungus Tolypocladium inflatum. The suggested nomenclature was proved feasible by naming introns present in mitogenomes of 16 fungi of different phyla, including both basal and higher fungal lineages although minor adjustment of the nomenclature is needed to fit certain special conditions. The nomenclature also had the potential to name plant/protist/animal mitochondrial introns. We hope future studies follow the proposed nomenclature to ensure direct comparison across different studies.

Comparison of the Mitochondrial Genome Sequences of Six Annulohypoxylon stygium Isolates Suggests Short Fragment Insertions as a Potential Factor Leading to Larger Genomic Size.

Mitochondrial DNA (mtDNA) is a core non-nuclear genetic material found in all eukaryotic organisms, the size of which varies extensively in the eumycota, even within species. In this study, mitochondrial genomes of six isolates of Annulohypoxylon stygium (Lév.) were assembled from raw reads from PacBio and Illumina sequencing. The diversity of genomic structures, conserved genes, intergenic regions and introns were analyzed and compared. Genome sizes ranged from 132 to 147 kb and contained the same sets of conserved protein-coding, tRNA and rRNA genes and shared the same gene arrangements and orientation. In addition, most intergenic regions were homogeneous and had similar sizes except for the region between cytochrome b (cob) and cytochrome c oxidase I (cox1) genes which ranged from 2,998 to 8,039 bp among the six isolates. Sixty-five intron insertion sites and 99 different introns were detected in these genomes. Each genome contained 45 or more introns, which varied in distribution and content. Introns from homologous insertion sites also showed high diversity in size, type and content. Comparison of introns at the same loci showed some complex introns, such as twintrons and ORF-less introns. There were 44 short fragment insertions detected within introns, intergenic regions, or as introns, some of them located at conserved domain regions of homing endonuclease genes. Insertions of short fragments such as small inverted repeats might affect or hinder the movement of introns, and these allowed for intron accumulation in the mitochondrial genomes analyzed, and enlarged their size. This study showed that the evolution of fungal mitochondrial introns is complex, and the results suggest short fragment insertions as a potential factor leading to larger mitochondrial genomes in A. stygium.

Characterization and Comparative Analysis of Olfactory Receptor Co-Receptor Orco Orthologs Among Five Mirid Bug Species.

The phytophagous mirid bugs of Apolygus lucorum, Lygus pratensis as well as three Adelphocoris spp., including Adelphocoris lineolatus, A. suturalis, and A. fasciaticollis are major pests of multiple agricultural crops in China, which have distinct geographical distribution and occurrence ranges. Like many insect species, these bugs heavily rely on olfactory cues to search preferred host plants, thereby investigation on functional co-evolution and divergence of olfactory genes seems to be necessary and is of great interest. In the odorant detection pathway, olfactory receptor co-receptor (Orco) plays critical role in the perception of odors. In this study, we identified the full-length cDNA sequences encoding three putative Orcos (AsutOrco, AfasOrco, and LpraOrco) in bug species of A. suturalis, A. fasciaticollis, and L. pratensis based on homology cloning method. Next, sequence alignment, membrane topology and gene structure analysis showed that these three Orco orthologs together with previously reported AlinOrco and AlucOrco shared high amino acid identities and similar topology structure, but had different gene structure especially at the length and insertion sites of introns. Furthermore, the evolutional estimation on the ratios of non-synonymous to synonymous (Ka/Ks) revealed that Orco genes were under strong purifying selection, but the degrees of variation were significant different between genera. The results of quantitative real-time PCR experiments showed that these five Orco genes had a similar antennae-biased tissue expression pattern. Taking these data together, it is thought that Orco genes in the mirid species could share conserved olfaction roles but had different evolution rates. These findings would lay a foundation to further investigate the molecular mechanisms of evolutionary interactions between mirid bugs and their host plants, which might in turn contribute to the development of pest management strategy for mirid bugs.

Heterogeneity of intron presence/absence in Olifantiella sp. (Bacillariophyta) contributes to the understanding of intron loss.

Although hypotheses have been proposed and developed to interpret the origins and functions of introns, substantial controversies remain about the mechanism of intron evolution. The availability of introns in the intermediate state is quite helpful for resolving this debate. In this study, a new strain of diatom (denominated as DB21-1) was isolated and identified as Olifantiella sp., which possesses multiple types of 18S rDNAs (obtained from genomic DNA; lengths ranged from 2,056bp to 2,988bp). Based on alignments between 18S rDNAs and 18S rRNA (obtained from cDNA; 1,783bp), seven intron insertion sites (IISs) located in the 18S rDNA were identified, each of which displayed the polymorphism of intron presence/absence. Specific primers around each IIS were designed to amplify the introns and the results indicated that introns in the same IIS varied in lengths, while terminal sequences were conserved. Our study showed that the process of intron loss happens via a series of successive steps, and each step could derive corresponding introns under intermediate states. Moreover, these results indicate that the mechanism of genomic deletion that occurs at DNA level can also lead to exact intron loss.

Plastomes of the green algae Hydrodictyon reticulatum and Pediastrum duplex (Sphaeropleales, Chlorophyceae).

BackgroundComparative studies of chloroplast genomes (plastomes) across the Chlorophyceae are revealing dynamic patterns of size variation, gene content, and genome rearrangements. Phylogenomic analyses are improving resolution of relationships, and uncovering novel lineages as new plastomes continue to be characterized. To gain further insight into the evolution of the chlorophyte plastome and increase the number of representative plastomes for the Sphaeropleales, this study presents two fully sequenced plastomes from the green algal family Hydrodictyaceae (Sphaeropleales, Chlorophyceae), one from Hydrodictyon reticulatum and the other from Pediastrum duplex.MethodsGenomic DNA from Hydrodictyon reticulatum and Pediastrum duplex was subjected to Illumina paired-end sequencing and the complete plastomes were assembled for each. Plastome size and gene content were characterized and compared with other plastomes from the Sphaeropleales. Homology searches using BLASTX were used to characterize introns and open reading frames (orfs) ≥ 300 bp. A phylogenetic analysis of gene order across the Sphaeropleales was performed.ResultsThe plastome of Hydrodictyon reticulatum is 225,641 bp and Pediastrum duplex is 232,554 bp. The plastome structure and gene order of H. reticulatum and P. duplex are more similar to each other than to other members of the Sphaeropleales. Numerous unique open reading frames are found in both plastomes and the plastome of P. duplex contains putative viral protein genes, not found in other Sphaeropleales plastomes. Gene order analyses support the monophyly of the Hydrodictyaceae and their sister relationship to the Neochloridaceae.DiscussionThe complete plastomes of Hydrodictyon reticulatum and Pediastrum duplex, representing the largest of the Sphaeropleales sequenced thus far, once again highlight the variability in size, architecture, gene order and content across the Chlorophyceae. Novel intron insertion sites and unique orfs indicate recent, independent invasions into each plastome, a hypothesis testable with an expanded plastome investigation within the Hydrodictyaceae.

Proliferation of group II introns in the chloroplast genome of the green alga Oedocladium carolinianum (Chlorophyceae).

BackgroundThe chloroplast genome sustained extensive changes in architecture during the evolution of the Chlorophyceae, a morphologically and ecologically diverse class of green algae belonging to the Chlorophyta; however, the forces driving these changes are poorly understood. The five orders recognized in the Chlorophyceae form two major clades: the CS clade consisting of the Chlamydomonadales and Sphaeropleales, and the OCC clade consisting of the Oedogoniales, Chaetophorales, and Chaetopeltidales. In the OCC clade, considerable variations in chloroplast DNA (cpDNA) structure, size, gene order, and intron content have been observed. The large inverted repeat (IR), an ancestral feature characteristic of most green plants, is present in Oedogonium cardiacum (Oedogoniales) but is lacking in the examined members of the Chaetophorales and Chaetopeltidales. Remarkably, the Oedogonium 35.5-kb IR houses genes that were putatively acquired through horizontal DNA transfer. To better understand the dynamics of chloroplast genome evolution in the Oedogoniales, we analyzed the cpDNA of a second representative of this order, Oedocladium carolinianum.MethodsThe Oedocladium cpDNA was sequenced and annotated. The evolutionary distances separating Oedocladium and Oedogonium cpDNAs and two other pairs of chlorophycean cpDNAs were estimated using a 61-gene data set. Phylogenetic analysis of an alignment of group IIA introns from members of the OCC clade was performed. Secondary structures and insertion sites of oedogonialean group IIA introns were analyzed.ResultsThe 204,438-bp Oedocladium genome is 7.9 kb larger than the Oedogonium genome, but its repertoire of conserved genes is remarkably similar and gene order differs by only one reversal. Although the 23.7-kb IR is missing the putative foreign genes found in Oedogonium, it contains sequences coding for a putative phage or bacterial DNA primase and a hypothetical protein. Intergenic sequences are 1.5-fold longer and dispersed repeats are more abundant, but a smaller fraction of the Oedocladium genome is occupied by introns. Six additional group II introns are present, five of which lack ORFs and carry highly similar sequences to that of the ORF-less IIA intron shared with Oedogonium. Secondary structure analysis of the group IIA introns disclosed marked differences in the exon-binding sites; however, each intron showed perfect or nearly perfect base pairing interactions with its target site.DiscussionOur results suggest that chloroplast genes rearrange more slowly in the Oedogoniales than in the Chaetophorales and raise questions as to what was the nature of the foreign coding sequences in the IR of the common ancestor of the Oedogoniales. They provide the first evidence for intragenomic proliferation of group IIA introns in the Viridiplantae, revealing that intron spread in the Oedocladium lineage likely occurred by retrohoming after sequence divergence of the exon-binding sites.

Host Factors Influencing the Retrohoming Pathway of Group II Intron RmInt1, Which Has an Intron-Encoded Protein Naturally Devoid of Endonuclease Activity.

Bacterial group II introns are self-splicing catalytic RNAs and mobile retroelements that have an open reading frame encoding an intron-encoded protein (IEP) with reverse transcriptase (RT) and RNA splicing or maturase activity. Some IEPs carry a DNA endonuclease (En) domain, which is required to cleave the bottom strand downstream from the intron-insertion site for target DNA-primed reverse transcription (TPRT) of the inserted intron RNA. Host factors complete the insertion of the intron. By contrast, the major retrohoming pathway of introns with IEPs naturally lacking endonuclease activity, like the Sinorhizobium meliloti intron RmInt1, is thought to involve insertion of the intron RNA into the template for lagging strand DNA synthesis ahead of the replication fork, with possible use of the nascent strand to prime reverse transcription of the intron RNA. The host factors influencing the retrohoming pathway of such introns have not yet been described. Here, we identify key candidates likely to be involved in early and late steps of RmInt1 retrohoming. Some of these host factors are common to En+ group II intron retrohoming, but some have different functions. Our results also suggest that the retrohoming process of RmInt1 may be less dependent on the intracellular free Mg2+ concentration than those of other group II introns.

The diversity of mtDNA rns introns among strains of Ophiostoma piliferum, Ophiostoma pluriannulatum and related species.

BackgroundBased on previous studies, it was suspected that the mitochondrial rns gene within the Ophiostomatales is rich in introns. This study focused on a collection of strains representing Ophiostoma piliferum, Ophiostoma pluriannulatum and related species that cause blue-stain; these fungi colonize the sapwood of trees and impart a dark stain. This reduces the value of the lumber. The goal was to examine the mtDNA rns intron landscape for these important blue stain fungi in order to facilitate future annotation of mitochondrial genomes (mtDNA) and to potentially identify mtDNA introns that can encode homing endonucleases which may have applications in biotechnology.ResultsComparative sequence analysis identified five intron insertion sites among the ophiostomatoid fungi examined. Positions mS379 and mS952 harbor group II introns, the mS379 intron encodes a reverse transcriptase, and the mS952 intron encodes a potential homing endonuclease. Positions mS569, mS1224, and mS1247 have group I introns inserted and these encode intact or eroded homing endonuclease open reading frames (ORF). Phylogenetic analysis of the intron ORFs showed that they can be found in the same insertion site in closely and distantly related species.ConclusionsBased on the molecular markers examined (rDNA internal transcribed spacers and rns introns), strains representing O. pilifera, O. pluriannulatum and Ophiostoma novae-zelandiae could not be resolved. Phylogenetic studies suggest that introns are gained and lost and that horizontal transfer could explain the presence of related intron in distantly related fungi. With regard to the mS379 group II intron, this study shows that mitochondrial group II introns and their reverse transcriptases may also follow the life cycle previously proposed for group I introns and their homing endonucleases. This consists of intron invasion, decay of intron ORF, loss of intron, and possible reinvasion.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-016-3076-6) contains supplementary material, which is available to authorized users.

Comparative and evolutionary analysis of α-amylase gene across monocots and dicots

α-amylase is an important enzyme involved in starch degradation to provide energy to the germinating seedling. The present study was conducted to reveal structural and functional evolution of this gene among higher plants. Discounting polyploidy, most plant species showed only a single copy of the gene making multiple isoforms in different tissues and developmental stages. Genomic length of the gene ranged from 1472bp in wheat to 2369bp in soybean, and the size variation was mainly due to differences in the number and size of introns. In spite of this variation, the intron phase distribution and insertion sites were mostly conserved. The predicted protein size ranged from 414 amino acid (aa) in soybean to 449aa in Brachypodium. Overall, the protein sequence similarity among orthologs ranged from 56.4 to 97.4%. Key motifs and domains along with their relative distances were conserved among plants although several species, genera, and class specific motifs were identified. The glycosyl hydrolase superfamily domain length varied from 342aa in soybean to 384aa in maize and sorghum while length of the C-terminal β-sheet domain was highly conserved with 61aa in all monocots and Arabidopsis but was 59aa in soybean and Medicago. Compared to rice, 3D structure of the proteins showed 89.8 to 91.3% similarity among the monocots and 72.7 to 75.8% among the dicots. Sequence and relative location of the five key aa required for the ligand binding were highly conserved in all species except rice.

Improvement of HIV-1 and Human T Cell Lymphotropic Virus Type 1 Replication-Dependent Vectors via Optimization of Reporter Gene Reconstitution and Modification with Intronic Short Hairpin RNA.

Cell-to-cell transmission is an efficient mechanism to disseminate human immunodeficiency virus type 1 (HIV-1) and human T cell lymphotropic virus type 1 (HTLV-1). However, it has been challenging to quantify the level of cell-to-cell transmission because the virus-producing cells cannot be easily distinguished from infected target cells. We have previously described replication-dependent vectors that can quantify infection events in cocultured cells. These vectors contain an antisense-oriented promoter and reporter gene interrupted by a sense-oriented intron from the human gamma-globin gene. This strategy prevents expression of the reporter gene in the transfected cells but permits its expression in target cells after infection. However, the gamma-globin intron is not efficiently removed by splicing in the aforementioned vectors, thereby reducing the level of reporter gene expression after transduction into target cells. Here, we used two approaches to improve the replication-dependent vectors. First, we improved the splicing events that remove the gamma-globin intron by optimizing the intron insertion site within the reporter gene. Second, we improved the packaging of the spliced RNA without the gamma-globin intron by targeting the intron-containing RNA via microRNA 30 (miR30)-based short hairpin RNAs. Using two optimized fluorescent reporter vectors and flow cytometry, we determined that multiply HIV-1-infected cells were generated at a higher frequency in coculture than in cell-free infection; furthermore, this increase was dependent upon viruses bearing HIV-1 Env. Compared with previously described vectors, these improved vectors can quantify the infection in lymphocytes and in primary cells with a higher sensitivity and allow the detection and quantitation of multiply infected cells, providing better tools to study retroviral cell-mediated infection. The human-pathogenic retroviruses HTLV-1 and HIV-1 can be transmitted more efficiently in vivo via direct contact of infected cells with healthy target cells than through cell-free virion-mediated infection. Despite its importance, cell-to-cell transmission has been difficult to quantify because the previously infected cells and the newly infected cells are mixed together in the same culture. In the current study, we generated vectors that are significantly improved over the previously described replication-dependent vectors. As a result, these improved vectors can efficiently detect and quantify cell-to-cell transmission or new infection events in cells in mixed culture. These luciferase- or fluorescence protein-based reporter vectors can be used to quantify and study HIV-1 or HTLV-1 cell-mediated infection in a simple one-step transfection/infection assay.

Cloning of genomic sequences of three crustacean hyperglycemic hormone superfamily genes and elucidation of their roles of regulating insulin-like androgenic gland hormone gene

The insulin-like androgenic gland hormone (IAG) gene in crustaceans plays an important role in male sexual differentiation, metabolism, and growth. However, the upstream regulation of IAG signaling schemes remains poorly studied. In the present study, we cloned the 5' flanking sequence of IAG and full-length genomic sequences of gonad-inhibiting hormone (Mn-GIH), molt-inhibiting hormone (Mn-MIH) and crustacean hyperglycemic hormone (Mn-CHH) in Macrobrachium nipponense. We identified the transcription factor-binding sites in the 5′ flanking sequence of IAG and investigated the exon–intron patterns of the three CHH superfamily genes. Each CHH superfamily gene consisted of two introns separating three exons. Mn-GIH and Mn-MIH shared the same intron insertion sites, which differed from Mn-CHH. We provided DNA-level evidence for the type definition. We also identified two cAMP response elements in the 5′ untranslated region. We further investigated the regulatory relationships between Mn-GIH, Mn-MIH, and Mn-CHH and IAG at the transcriptional level by injection of double-stranded RNA (dsRNA). IAG transcription levels were significantly increased to 660.2%, 472.9%, and 112.4% of control levels in the Mn-GIH dsRNA, Mn-MIH dsRNA, and Mn-CHH dsRNA groups, respectively. The results clearly demonstrated that Mn-GIH and Mn-MIH, but not Mn-CHH, negatively regulate the expression of the IAG gene.

I-OmiI and I-OmiII: Two intron-encoded homing endonucleases within the Ophiostoma minus rns gene

The mitochondrial small subunit ribosomal RNA (rns) gene of the ascomycetous fungus Ophiostoma minus [strain WIN(M)371] was found to contain a group IC2 and a group IIB1 intron at positions mS569 and mS952 respectively. Both introns have open reading frames (ORFs) embedded that encode double motif LAGLIDADG homing endonucleases (I-OmiI and I-OmiII respectively). Codon-optimized versions of I-OmiI and I-OmiII were synthesized for overexpression in Escherichia coli. The in vitro characterization of I-OmiII showed that it is a functional homing endonuclease that cleaves the rns target site two nucleotides upstream (sense strand) of the intron insertion site generating 4 nucleotide 3′ overhangs. The endonuclease activity of I-OmiII was tested using linear and circular substrates and cleavage activity was evaluated at various temperatures. The I-OmiI protein was expressed in E. coli, but purification was difficult, thus the endonuclease activity of this protein was tested via in vivo assays. Overall this study showed that there are many native forms of functional homing endonucleases yet to be discovered among fungal mtDNA genomes.

Two-Gene Phylogeny of Bright-Spored Myxomycetes (Slime Moulds, Superorder Lucisporidia)

Myxomycetes, or plasmodial slime-moulds, are one of the largest groups in phylum Amoebozoa. Nonetheless, only ∼10% are in the database for the small subunit (SSU) ribosomal RNA gene, the most widely used gene for phylogenetics and barcoding. Most sequences belong to dark-spored Myxomycetes (order Fuscisporida); the 318 species of superorder Lucisporidia (bright-spored) are represented by only eleven genuine sequences. To compensate for this, we provide 66 new sequences, 37 SSU rRNA and 29 elongation factor 1-alpha (EF-1α), for 82% of the genera of Lucisporidia. Phylogenetic analyses of single- and two-gene alignments produce congruent topologies and reveal both morphological characters that have been overemphasised and those that have been overlooked in past classifications. Both classical orders, Liceida and Trichiida, and several families and genera are para/polyphyletic; some previously unrecognised clades emerge. We discuss possible evolutionary pathways. Our study fills a gap in the phylogeny of Amoebozoa and provides an extensive SSU rRNA sequence reference database for environmental sampling and barcoding. We report a new group I intron insertion site for Myxomycetes in one Licea.